Methods and Compositions for Producing Extracellular Matrix

Abstract

Embodiments herein include methods, kits, and compositions for producing extracellular matrix (ECM). In some embodiments, the methods include differentiating fibroblasts into induced pluripotent stem cells, expanding the induced pluripotent stem cells, and differentiating the induced pluripotent stem cells into fibroblasts. The fibroblasts can produce mature ECM, which can be isolated and used for medical and / or cosmetic products and medical and / or cosmetic procedures.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority under PCT Rule 4.10 to U.S. National Application No. 15 / 662901, filed Jul. 28, 2017, entitled "Methods and Compositions for Manufacturing Extracellular Matrix", which is hereby incorporated by reference in its entirety.

[0002] Reference to a Sequence Listing, Table, or Computer Program Listing This application is filed together with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BREK001WO.TXT, created on Jun. 22, 2018 and last updated, with a size of 247,550 bytes. The information in the electronic format of the Sequence Listing is hereby incorporated by reference in its entirety.

[0003] Field Some embodiments of the present specification relate to methods and compositions for manufacturing extracellular matrix (ECM). In some embodiments, methods and compositions for cell culture are described.

Background Art

[0004] The extracellular matrix (ECM) is a versatile biomaterial with many cosmetic and therapeutic applications. Most of the connective tissue consists of collagen, and to a much lesser extent (based on relative abundance by mass), other glycoproteins such as laminin, fibronectin, and glycosaminoglycans (GAGs) (including hyaluronic acid and other sulfated GAGs such as aggrecan and perlecan). However, collagen is cross-linked and requires enzymatic or chemical degradation to isolate and manufacture into useful products for human use. Examples include the use of bovine and porcine dermis, porcine small intestinal submucosa, and human cadaver-derived tissues such as skin and bone after pepsin digestion or chemical modification, all of which are well known in the art.

[0005] Animal sources of ECM such as porcine and bovine tissues carry the risk of unwanted immune reactions, including known allergies to bovine and porcine antigens or allergens, mainly proteins, but human cells using animal-derived components (most often containing bovine serum, bovine albumin, or porcine trypsin), or plant-derived proteins other than human (including soybean trypsin inhibitor produced in plants or recombinant human albumin that may contain residual plant proteins or polypeptides) are also subject to the same risk. Thus, the commercial utility of ECM from animal sources can be limited by concerns about safety and / or regulatory hurdles to demonstrate removal of animal components to safer residual levels required for approval for commercial use. The human immune system is highly sensitive and can react to extremely small amounts of antigen, and animal and plant proteins have been shown to cause unwanted immune reactions, including allergic reactions that can potentially be life-threatening and in some cases cause death. Animal-derived and plant-derived protein components, as well as human cells grown in animal-derived or plant-derived protein components, are collectively referred to as "xeno." On the other hand, products manufactured without contact with these animal-derived and plant-derived protein components are known as "xeno-free." Conventional approaches to manufacturing ECM in xeno media may require removal of animal-derived and / or plant-derived protein components, limiting the commercial utility of these methods.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

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Non-Patent Documents

[0007] [Non-Patent Document 1] Warren et al. (2010), Cell Stem Cell 7: 618 - 30 [Non-Patent Document 2] Moon et al., (2011), Cell Res, 21: 1305 - 15 [Non-Patent Document 3] Hou et al., (2013), Science 341: 651 - 654 [Non-Patent Document 4] Ye et al., (2016), Cell Research 26: 34 - 35 [Non-Patent Document 5] Goodpaster et al., (2008), J. Histochem Cyotchem, 56: 347 - 58 [Non-Patent Document 6] Xu et al., Scientific Reports 5: 8480 DOI: 10.1038 / srep08480 [Non-Patent Document 7] Sambrook et al., "Molecular Cloning: A Laboratory Manual (3rd Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2000 [Non-Patent Document 8] Pinney E, (2011) International Journal of Stem Cells 4: 70 - 75 [Non-Patent Document 9] Menen et al., (2012) Anticancer Research 32: 1573 - 1578 [Summary of the Invention] [Problems to be Solved by the Invention]

[0008] Human ECM may be derived from allogeneic tissues from cadavers. However, such cadaver-derived ECM poses a risk of disease transmission, and each donor has a limited amount of human ECM that can be obtained (e.g., a 70 kg human contains less than 20% by mass of human collagen or less than a few kilograms of ECM raw material), resulting in limitations in commercial scalability. Furthermore, cadaver-derived tissues involve costly safety tests to reduce some of the risks of disease transmission and must be extensively tested for each cadaver donor for pathogens that cause some diseases, including viruses and bacteria.

[0009] Furthermore, ECM may be used in animal feed. However, in practice, conventional approaches to producing ECM using cell culture or collection from cadavers can be prohibitively expensive on a commercial scale. Described herein according to some embodiments are methods, compositions, and kits for efficiently producing ECM on a commercial scale while minimizing the risks of disease transmission and immunogenicity.

Means for Solving the Problems

[0010] Some embodiments include a method of manufacturing an extracellular matrix. The method may include differentiating induced pluripotent stem cells (iPSCs) into production fibroblasts. The method may include culturing the production fibroblasts, by which the production fibroblasts can produce an extracellular matrix (ECM). The method may include isolating the ECM from the production fibroblasts and thus manufacturing the ECM. In some embodiments, the method further includes dedifferentiating precursor fibroblasts to form iPSCs prior to differentiating the iPSCs into production fibroblasts. In some embodiments, the method further includes expanding the iPSCs, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30-fold (including the range between any two of the recited values) prior to differentiation. In some embodiments, the method further includes constructing an iPSC bank prior to differentiation. In some embodiments, only iPSCs are differentiated from a single donor. In some embodiments, the step of culturing the production fibroblasts is in normoxia. In some embodiments, the step of culturing the production fibroblasts does not include culturing mesenchymal stem cells (MSCs). In some embodiments, the precursor fibroblasts include adult dermal (biopsy) fibroblasts. In some embodiments, the step of isolating the ECM includes purifying the ECM, thereby manufacturing a composition that is at least about 80 w / w% ECM. In some embodiments, the step of purifying the ECM includes washing the ECM in an acidic buffer and contacting a solution containing the production fibroblasts and the ECM with dextranase. In some embodiments, the step of isolating the ECM includes separating the ECM from a substrate or solid phase. In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). In some embodiments, the isolated ECM is not digested by any protease and is thus intact.In some embodiments, the ECM comprises collagen. In some embodiments, about 90% (w / w) of the ECM is COL1, and about 10% is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof. In some embodiments, the method further comprises contacting a progenitor fibroblast with a dedifferentiation factor, thereby dedifferentiating the progenitor fibroblast into an iPSC. In some embodiments, the iPSC has no viral insertions encoding Oct family members, Sox family members, Klf family members. In some embodiments, the iPSC is footprint free. In some embodiments, the method does not include any of embryonic stem (ES) cells, bone marrow multipotent stem cells, ES-derived MSCs, or non-pluripotent neonatal foreskin fibroblast cell lines. In some embodiments, the ECM comprises the c-terminal propeptide of COL1, or triple helix or non-reducing gamma-type fibrillar collagen, or both. In some embodiments, the ECM comprises triple helix and / or non-reducing gamma-type fibrillar collagen. In some embodiments, the method further comprises contacting the producing fibroblast with serum until the producing fibroblast produces mature collagen, and then gradually reducing the amount of serum until there is at least a 95% reduction in serum concentration. In some embodiments, the gradual reduction occurs over at least about 5 days.

[0011] Some embodiments include a kit for manufacturing an ECM. The kit may include a composition containing human fibroblasts. The kit may include a dedifferentiation factor. The kit may include a fibroblast differentiation factor. In some embodiments, all of the fibroblasts in the composition are from a single donor. In some embodiments, the kit further includes a substrate such as dextran microcarriers. In some embodiments, the kit further includes dextranase. In some embodiments, the kit further includes dextranase and DNAase.

[0012] Some embodiments include a composition comprising at least 80% (w / w) extracellular matrix, wherein the extracellular matrix is produced according to any of the above methods.

[0013] Some embodiments include a cell culture comprising iPSC-derived fibroblasts that produce a mature extracellular matrix. The cell culture may further comprise dedifferentiation factors. In some embodiments, at least 50% (w / w) of the composition comprises ECM.

[0014] Some embodiments include a method of manufacturing an extracellular matrix (ECM). The method may include culturing fibroblasts and / or mesenchymal stem cells (MSCs) on a substrate. The substrate may include at least two surfaces. The culture may be serum-free and xenofree. The culture may be performed until the fibroblasts and / or MSCs define a three-dimensional shape on at least two surfaces and until at least 80% of the fibroblasts and / or MSCs arrest their cell cycle. The method may then include contacting the fibroblasts and / or MSCs with serum for at least about two weeks, through which the fibroblasts and / or MSCs produce a soluble mature ECM. In this way, a solution containing the soluble mature ECM and fibroblasts or MSCs, where the solution is xenofree, can be produced. The method may further include isolating the soluble mature ECM from the producing fibroblasts, thus manufacturing an ECM that is a mature xenofree ECM. In some embodiments, the step of isolating the ECM includes separating the ECM from the substrate. In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). In some embodiments, the isolated ECM is not digested by any protease and is thus intact. In some embodiments, the method further includes expanding a human pluripotent cell culture, where the expansion is serum-free and xenofree, thereby producing human pluripotent cells, and contacting the human pluripotent cells with a differentiation factor, through which the human pluripotent cells differentiate into fibroblasts or MSCs. In some embodiments, the contact between the fibroblasts and / or MSCs and the serum is from about two weeks to about eight weeks. In some embodiments, the contact between the fibroblasts and / or MSCs and the serum is at least about eight weeks. In some embodiments, the human pluripotent cells include induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are footprint-free. In some embodiments, the iPSCs are derived from a single donor. In some embodiments, the method further includes manufacturing a cosmetic composition comprising the mature xenofree ECM. In some embodiments, the method includes contacting with serum for at least It further includes the step of contacting fibroblasts or MSCs with ascorbic acid for two weeks. In some embodiments, fibroblasts or MSCs on at least two surfaces define a three-dimensional shape, and before at least 70% of the fibroblasts or MSCs arrest their cell cycle, the fibroblasts or MSCs are not contacted with serum. In some embodiments, the amount of serum is about 0.1% - 10% (v / v). In some embodiments, the amount of serum is about 1 - 2% (v / v). In some embodiments, the serum includes clinically graded fetal bovine serum, pooled human serum, or a combination thereof. In some embodiments, the pluripotent cells are cell lines previously grown using animal components. In some embodiments, the mature xenofree ECM includes fibrillar collagen. In some embodiments, the mature xenofree ECM includes the c-terminal propeptide of COL1, or triple helix or non-reducing gamma-type fibrillar collagen, or both. In some embodiments, the ECM includes triple helix and / or non-reducing gamma-type fibrillar collagen. In some embodiments, the solution includes at least 250 μg of collagen per 1 cm 2 of the substrate. In some embodiments, the produced mature xenofree ECM includes at least 250 μg of collagen per 1 cm 2 of the substrate. In some embodiments, the pluripotent cells are derived from a single donor. In some embodiments, the method further includes the step of detecting the amount of mature ECM in the solution. In some embodiments, the method further includes the step of collecting a certain amount of used culture medium from the solution and isolating soluble mature ECM from the used culture medium.

[0015] Some embodiments include a solution containing fibroblasts or MSCs and soluble mature ECM produced according to any of the methods of the above paragraphs. The solution may be xenofree, and the soluble mature ECM may include cross-linked collagen.

[0016] Some embodiments include a method of manufacturing an extracellular matrix (ECM). The method may include providing fibroblasts in a medium containing a certain concentration of serum. The method may include gradually reducing the amount of serum in the medium containing fibroblasts until the medium contains a serum concentration of 5% or less. The method may include culturing the fibroblasts for at least about two weeks after the gradual reduction of serum, through which the fibroblasts produce a soluble ECM, thereby producing a solution containing fibroblasts and the soluble ECM. The method may include isolating the soluble ECM from the fibroblasts, thereby manufacturing the ECM. In some embodiments, the step of isolating the ECM includes separating the ECM from a substrate or solid phase. In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). In some embodiments, the isolated ECM is not digested by any protease and is thus intact. In some embodiments, the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is at least 0.7 times the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. In some embodiments, the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is at least 0.9 times the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. In some embodiments, the step of gradually reducing the amount of serum is performed without cell expansion or cell subculture. In some embodiments, the serum is gradually reduced for at least about five days. In some embodiments, at least about 90% of the fibroblasts in the solution are in the G0 cell cycle stage. In some embodiments, less than 1% of the fibroblasts in the solution are undergoing apoptosis. In some embodiments, the solution is a nanostructure containing the soluble ECM and includes nanostructures having a maximum diameter of 200 nm to 10,000 nm. In some embodiments, the step of manufacturing the ECM is performed without sterile filtration (so as not to exclude the nanostructures). In some embodiments, the step of isolating the soluble ECM from the fibroblasts is performed without sterile filtration.

[0017] Some embodiments include a solution comprising fibroblasts and a soluble ECM. At least about 90% of the fibroblasts in the solution may be in the G0 cell cycle stage. Less than 1% of the fibroblasts in the solution may be undergoing apoptosis. The solution may include nanostructures comprising the soluble ECM, and may include nanostructures having a maximum diameter of 200 nm to 10,000 nm. In some embodiments, the soluble ECM is produced by the method according to any one of claims 48 to 57. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

Figure 1

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Figure 3

[0019] ECM may be useful for some cosmetic and therapeutic applications, but conventional methods of producing ECM may have issues related to scalability, ease of use, contamination (including immunogenicity in human hosts), disease transmission, and cost effectiveness. Described herein are methods, compositions, and kits useful for efficiently producing ECM, including on a commercial scale. ECM according to the methods, compositions, and kits of some embodiments herein may include relatively high levels of commercially useful mature collagen (such as triple helix or non-reducing gamma-type fibrillar collagen, or both, etc.), and may potentially have relatively few contaminants such as potentially harmful foreign substances.

[0020] Some embodiments include methods, compositions, and kits for creating non-embryonic human fibroblasts for use as a bioreactor for manufacturing human extracellular matrix (ECM). According to these embodiments, mature fibroblasts can be dedifferentiated into induced pluripotent stem cells (iPSCs), expanded, and redifferentiated into mature non-embryonic producing fibroblasts. These producing fibroblasts can be used to manufacture collagen-rich ECM.

[0021] Some embodiments include methods, compositions, and / or kits for manufacturing mature xenofree ECM. According to these embodiments, cells can be cultured for extended periods (e.g., up to 8 weeks, e.g., over 2 - 3 weeks or more), resulting in an ECM having desired properties such as increased cross-linking of collagen and excellent ECM solubility. In contrast, many conventional approaches for manufacturing ECM can perform cell culture in much shorter times, e.g., 12 - 17 days. Surprisingly, cell cultures of 2 - 3 weeks or more according to some embodiments herein produce an ECM with increased cross-linking and solubility compared to shorter-term cultures.

[0022] Some embodiments include a method of culturing fibroblasts for producing an extracellular matrix (ECM) in which the fibroblasts are gradually detached from serum. According to these embodiments, the fibroblasts can first be cultured in a serum-containing formulation, and the amount of serum can then be gradually reduced (e.g., by removing and replacing the culture medium to lower the serum content and / or transferring the cells to a different culture medium with a lower serum content). The cells can produce mature soluble ECM for at least two weeks after the reduction of serum, and the soluble ECM can be isolated from the cells. "Soluble" (e.g., in the context of soluble ECM) is used herein according to its ordinary meaning in the art and includes the type or fraction of ECM that is dissolved or can be dissolved in the aqueous phase. Thus, the soluble ECM can be recovered and maintained in the aqueous phase. In some embodiments, the soluble ECM does not precipitate in the aqueous phase. In some embodiments, the soluble ECM can be stably maintained in the aqueous phase for at least 24 hours under the same conditions with minimal precipitation, e.g., about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less of the soluble ECM precipitate. This method can advantageously result in highly soluble ECM that can be useful in cosmetics and pharmaceuticals. In some embodiments, the ECM is efficiently produced without any cell expansion or subculturing. It is noted that in some embodiments, both soluble and insoluble ECM can be recovered from the same culture, and advantageously, a higher yield can be obtained by recovering ECM from both phases than by obtaining ECM from only one phase.

[0023] Extracellular matrix (ECM) "Extracellular matrix" (ECM) is used herein according to its ordinary meaning in the art and includes molecules secreted by cells, such as proteins and carbohydrates, that provide a structure to support cells. The ECM may include fibrous proteins such as collagen. Human ECM may include several collagen proteins, for example, COL1, COL3, COL4, COL5, and COL6, and combinations of these proteins. Exemplary polypeptide sequences of Homo sapiens COL1, COL3, COL4, COL5, and COL6 are shown in Table 1 below. In some embodiments of the methods, compositions, and / or kits, the cell culture produces ECM that can be isolated from the cell culture. In some embodiments, the ECM consists essentially of or consists of human ECM, including human ECM. In some embodiments, the step of isolating the ECM from the cell culture includes the step of purifying the ECM. In this way, a human ECM product consisting essentially of or consisting of the isolated ECM can be produced.

[0024]

Table 1-1

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Table 1-16

[0025] The fibrous collagenous ECM collagen, a type of mature ECM by the methods, compositions, and kits of some embodiments of the present specification, may contain the main component COL1, and may also contain COL3, COL4, COL5, and / or COL6 (for example, about 90% COL1 and about 10% COL3, COL4, COL5, and / or COL6), but other ratios of (i) COL1 and (ii) COL3, COL4, COL5, and / or COL6 are appropriate (for example, about 97% and 3% respectively, about 95% and 5% respectively, about 93% and 7% respectively, about 85% and 15% respectively, about 80% and 20% respectively, about 75% and 25% respectively, or about 70% and 30% respectively).

[0026] ECM, and in particular, human ECM products when produced by the methods, compositions, and kits of some embodiments herein, are useful for treating tissues of patients suffering from musculoskeletal disorders, orthopedic dysfunctions and associated pain, cardiovascular disorders, skin diseases, cosmetic skin and hair conditions associated with aging that require improvement in appearance, solid tumors that require treatment including surgical wounds, surgical resection, chemotherapy, and immunotherapy. Use of human ECM products produced by the methods, kits, and compositions according to some embodiments herein include, by way of example, osteochondral defect repair, osteoarthritis, degenerative disc disease, orthopedic surgical wounds; surgical wounds with tumor resection cavities, cardiovascular regeneration devices and biologics, skin wound devices and biologics, skin fillers for treating wrinkles, topical cosmetics, therapeutic hair growth, and cell delivery and drug delivery vehicles for increased persistence and reduced unwanted immune responses when transplanted into a patient, including medical devices and biologics for musculoskeletal applications. In some embodiments, ECM (e.g., human ECM products) produced by the methods, compositions, and kits of some embodiments herein are used in at least one of a medical product, cosmetic, drug, medical device, treatment, or biologic. In some embodiments, ECM can be used in at least one of a medical product, biologic, medical device, drug, or any other product or composition regulated by the FDA. In some embodiments, ECM can be used in at least one of a medical or cosmetic procedure.

[0027] The use of human cell cultures for the production of human ECM by the methods, compositions, and kits of some embodiments of this specification can provide advantages over animal- and cadaver-derived ECM. For example, in some embodiments, the cells in culture may be from a single donor, can be easily expanded, and / or may be xenofree. On the other hand, conventional approaches for producing ECM by cell culture present challenges, such as those related to commercial scale, cost-effectiveness, and the presence of non-human components. For example, fibroblast-derived human ECM and manufacturing methods from cell culture have been described. For example, the patent group by Naughton et al. (referred to as the "Naughton patents," U.S. Patent Nos. 6,378,527; 5,830,708; 7,118,746; 6,372,494; 8,257,947; 8,530,415; 8,535,913; 9,034,312; 8,852,637; 8,128,924; 8,138,147; 8,361,485; 8,476,231; and 9,458,486, which are hereby incorporated by reference in their entirety) disclose various compositions and production methods of soluble and insoluble human ECM. For example, the Naughton patents disclose the use of animal proteins such as fetal bovine serum, calf serum, and porcine trypsin to support cell expansion and the production of human ECM. These processes using animal components, for example, in the production of CosmoDerm (trademark) and CosmoPlast (trademark) dermal filler products, also utilize porcine pepsin for collagen after the ECM is produced. For example, porcine carbohydrates such as porcine heparin may cause harmful immune reactions in humans.

[0028] The Naughton patent also discloses soybean trypsin inhibitor. Without being limited to theory, soybean trypsin inhibitor can be used in the absence of serum and can reduce harmful proteolysis in cell culture. However, some plant proteins, and particularly soybean trypsin inhibitor, can cause unwanted immune reactions in humans, and even potentially allergies or anaphylactic shock or rarely death. Therefore, it is considered that replacing enzyme inhibitors found in animal serum with plant components such as soybean trypsin inhibitor can still cause unwanted immune reactions. Furthermore, soybean trypsin inhibitor may have enzyme activities that can affect a composition containing soybean trypsin inhibitor, or skin tissue where soybean trypsin inhibitor is exposed, for example, upon topical or transdermal application. Soybean trypsin inhibitor has also been shown to inhibit hair growth. Removing soluble soybean trypsin inhibitor is formally possible, but doing so involves extensive and costly purification methods in an unclear mixture of cell secretory products in used culture medium, resulting in a product where the exact active ingredient may not be known, so its commercial utility is limited. Therefore, some embodiments do not include animal-derived products or soybean trypsin inhibitor. Some embodiments do not include animal-derived or plant-derived products.

[0029] Xenofree systems and methods can advantageously avoid the use of animal and / or plant-derived components from the start. Thus, a xenofree method of manufacturing ECM according to some embodiments herein can eliminate the need for clinical trials regarding the removal of animal (and / or plant)-derived components. For example, the methods and compositions according to some embodiments herein can produce soluble ECM compositions for cosmetic use and that do not necessarily contain known plant allergens such as soybean protein. Furthermore, ECM compositions produced according to some embodiments herein that do not contain animal or plant-derived components will not require safety warnings regarding potential allergies to animal products or plant products such as soybean protein.

[0030] In some embodiments, the cell-based in vitro culture method can produce xeno-free human ECM on a scale greater than one human per batch. Further, since the cell lines can be extensively tested, the xeno-free ECM may have a reduced risk of disease transmission, and since the tested cell lines can be expanded, each set of tests can support many commercial-scale batches of products manufactured using human ECM.

[0031] The fibroblast-conditioned medium may contain soluble ECM and, thus, can also be used for cosmetic purposes in some embodiments. Without being limited by theory, it has been observed herein that fetal and embryonic ECM contain relatively lower levels of maturation and non-reducing crosslinks. However, the soluble human ECM produced by some embodiments herein can contain a greater amount of mature type I collagen, such as a greater amount of mature triple helix type I collagen, as well as slightly less fibrillar collagen, such as types III, V, and VI collagen.

[0032] Several approaches can be used to assess the presence and / or level of mature collagen in the ECM according to the embodiments herein. For example, the amount of crosslinked mature collagen can be measured on a reducing SDS-PAGE gel using adult tissue-derived type I collagen for comparison.

[0033] Pluripotent cells "Pluripotent cells" are used herein according to their ordinary meaning in the art and include classes of cells that can differentiate according to multiple different fates. Examples of pluripotent cells include, but are not limited to, induced pluripotent stem cells (iPSCs) and embryonic stem (ES) cells. In some embodiments, the pluripotent cells suitable for the methods, compositions, and / or kits herein comprise, consist of, or consist essentially of iPSCs. In some embodiments, the pluripotent cells suitable for the methods, compositions, and kits herein comprise, consist of, or consist essentially of iPSCs or ES cells. In some embodiments, the pluripotent cells suitable for the methods, compositions, and kits herein comprise, consist of, or consist essentially of iPSCs, but not ES cells. In some embodiments, the pluripotent cells suitable for the methods, compositions, and / or kits herein are human cells. In some embodiments, the pluripotent cells suitable for the methods, compositions, and / or kits herein are derived from a single donor.

[0034] Without being limited by theory, pluripotent cells (either artificial or embryonic in origin) have not conventionally produced sufficient fibrillar collagenous ECM in cell culture. However, in the methods, compositions, and kits of some embodiments herein, pluripotent cells can provide a rich supply of cells from a single donor (which can be considered almost limitless) and can be used when differentiating pluripotent cells into fibroblasts that secrete and precipitate ECM. Thus, with the compositions, methods, and kits of some embodiments herein, the process of dedifferentiating fibroblasts into iPSCs, expanding the iPSCs, and then redifferentiating the iPSCs into fibroblasts can produce a commercial-scale quantity of single-donor fibroblasts for producing ECM. Since the cells are from a single donor, the uniformity of the produced ECM product can be greater and the risk of contamination and / or disease transmission can be lower. The process of generally producing iPSCs from fibroblasts and then redifferentiating the iPSCs back into fibroblasts is labor-intensive, but those skilled in the art will understand that the labor is worthwhile due to the practical advantages discussed herein for the practical applications discussed herein.

[0035] The term "induced pluripotent stem cell" (iPSC) is used herein according to its ordinary meaning in the art and includes a class of cells produced by the dedifferentiation of somatic cells into cells having characteristics similar to ES cells. Some approaches recognized in the art for making iPSCs are suitable for the methods, kits, and compositions of the embodiments herein. In some embodiments, iPSCs are made by contacting somatic cells with dedifferentiation factors. As described in detail herein, dedifferentiation factors can include nucleic acids, polypeptides, and / or small molecules that induce somatic cells to differentiate into iPSCs.

[0036] As used herein, the term "dedifferentiation factor" (including variations of this base term) refers to a set of gene products, and / or nucleic acids encoding gene products, and / or small molecules sufficient to dedifferentiate somatic cells (e.g., fibroblasts) into iPSCs. Dedifferentiation factors can be used to dedifferentiate somatic cells (e.g., fibroblasts) into iPSCs by the methods of some embodiments herein. Dedifferentiation factors can also be included with compositions and kits according to some embodiments herein since they can be useful for dedifferentiating somatic cells (e.g., fibroblasts) into iPSCs. In some embodiments, the dedifferentiation factor includes two or more transcription factors. In some embodiments, the dedifferentiation factor consists essentially of, or consists of, an Oct family member (e.g., Oct3 / 4 or POU5F1), a Sox family member (e.g., Sox1, Sox2, Sox3, Sox4, Sox11, or Sox15), a Klf family member (e.g., Klf1, Klf2, Klf4, or Klf5), and at least one of (i) a Myc family member (e.g., c-Myc, L-Myc, or N-Myc), (ii) Nanog, (iii) Lin28 or Lin28B, or (iv) Glis1, and / or (in the case of dedifferentiating fibroblasts) consists essentially of, or consists of, an Oct family member (e.g., Oct4) and Bmi1. In some embodiments, the dedifferentiation factor consists essentially of, or consists of, a chemical dedifferentiation factor (e.g., a small molecule).

[0037] In some embodiments, the dedifferentiation factors are provided as one or more nucleic acids encoding gene products sufficient to dedifferentiate somatic cells (e.g., fibroblasts) into iPSCs. Such dedifferentiation factors may be provided in a single vector, or in a series of more than one vector. Examples of suitable vectors for the methods, compositions, and kits of some embodiments include, but are not limited to, retroviral vectors, adenoviral vectors, adeno-associated vectors, lentiviral vectors, and the like. In some embodiments, each of the nucleic acids encoding the dedifferentiation factors is operably linked to a promoter (it is also contemplated that two or more nucleic acids may be separated by, for example, an IRES or 2A element and be under the control of the same promoter). In some embodiments, the dedifferentiation factors are provided as one or more gene products (e.g., proteins) sufficient to dedifferentiate somatic cells (e.g., fibroblasts into iPSCs). In some embodiments, the dedifferentiation factors are provided as a collection of proteins. In some embodiments, the dedifferentiation factors are provided as a single polypeptide that can be cleaved to yield individual dedifferentiation factors. The polypeptide may further include tags, such as nuclear localization sequences, that facilitate translocation and localization to appropriate parts of the target cell. In some embodiments, the dedifferentiation factors include the chemical dedifferentiation factors described herein. There may be additional advantages associated with "footprint-free" dedifferentiation factors, which do not use potentially harmful viruses to deliver factors that induce pluripotency and do not insert foreign substances that may increase the risk of insertional mutagenesis into the host genome (e.g., delivered by non-integrating vectors, vector removal, direct administration of mRNA or polypeptides, or chemical induction of pluripotency). Thus, in some embodiments, the dedifferentiation factors are footprint-free. Examples of footprint-free generation of iPSCs from somatic cells by delivery of mRNAs encoding Klf4, c-Myc, Oct4, and Sox2 to somatic cells can be found, for example, in Warren et al. (2010), Cell Stem Cell 7: 618-30, which is hereby incorporated by reference in its entirety In some embodiments, such mRNA encoding a dedifferentiation factor is used to create iPSCs. In some embodiments, chemical induction of pluripotency is as described herein.

[0038] Combinations of Oct family members (e.g., Oct3, Oct4, and / or POU5F1) and Sox family members (e.g., Sox1, Sox2, Sox3, Sox4, Sox11, or Sox15) have been reported to be sufficient to dedifferentiate somatic cells into iPSCs. See U.S. Patent No. 9,683,232, which is hereby incorporated by reference in its entirety. Thus, in some embodiments, the dedifferentiation factor consists essentially of or consists of an Oct family member and a Sox family member. See U.S. Patent No. 9,683,232. Further, without being limited by theory, the inclusion of additional factors is thought to enhance the efficiency of dedifferentiation. For example, in some embodiments, the dedifferentiation factor consists essentially of or consists of an Oct family member, a Klf family member (e.g., Klf1, Klf2, Klf4, or Klf5), and a Sox family member. For example, a combination of Oct3 / 4, Klf4, c-Myc, and Sox2 is sufficient to dedifferentiate somatic cells (and particularly fibroblasts) into iPSCs. See U.S. Patent No. 8,058,065, which is hereby incorporated by reference in its entirety. Accordingly, in some embodiments, the dedifferentiation factor consists essentially of or consists of Oct3 / 4, Klf4, c-Myc, and Sox2.

[0039] In some embodiments, the dedifferentiation factors consist essentially of or consist of Oct family members (e.g., Oct3, Oct4, or POU5F1), Klf family members (e.g., Klf1, Klf2, Klf4, or Klf5), Myc family members (e.g., c-Myc, L-Myc, or N-Myc), and Sox family members (e.g., Sox1, Sox2, Sox3, Sox4, Sox11, or Sox15). Further, combinations of Oct3 / 4, Klf4, Sox2, and at least one of (i) Myc family members, (ii) Nanog, (iii) Lin28 or Lin28B, or (iv) Glis1 have also been reported to be sufficient to dedifferentiate somatic cells, and in particular fibroblasts, into iPSCs. See US 9,447,408, which is hereby incorporated by reference in its entirety. Thus, in some embodiments, the dedifferentiation factors consist essentially of or consist of Oct3 / 4, Klf4, Sox2, and at least one of (i) Myc family members, (ii) Nanog, (iii) Lin28 or Lin28B, or (iv) Glis1.

[0040] In particular, fibroblasts have also been reported to be able to be reprogrammed into iPSCs using a combination of the factors Oct4 and Bmi1 (e.g., such that Bmi1 can substitute for a combination of Sox2, Klf4, and / or c-Myc). See Moon et al., (2011), Cell Res, 21: 1305-15, which is hereby incorporated by reference in its entirety. Thus, in some embodiments, if, for example, progenitor fibroblasts are to be dedifferentiated into iPSCs, the dedifferentiation factors consist essentially of or consist of Oct4 and Bmi1.

[0041] A cocktail of small molecules, "VC6TF" (V, VPA; C, CHIR99021 or CHIR; 6, 616452; T, tranylcypromine; F, forskolin) has been shown to be able to induce pluripotent stem cells from somatic cells. Hou et al., (2013), Science 341: 651-654, which is hereby incorporated by reference in its entirety. For example, addition of an inhibitor of the H3K79 histone methyltransferase DOT1L, EPZ 004777 (EPZ, E), and a retinoid acid receptor (RAR) agonist, Ch 55 to VC6TF has been shown to promote dedifferentiation of somatic cells. Ye et al., (2016), Cell Research 26: 34-35, which is hereby incorporated by reference in its entirety. Thus, in some embodiments, the dedifferentiation factor consists of, consists essentially of, or comprises a chemical dedifferentiation factor, such as VC6TF, or EPZ 004777, DOT1L, and Ch 55 together with VC6TF.

[0042] In some embodiments, the iPSC is derived from somatic cells of a single donor. Without being limited by theory, iPSCs (or other pluripotent cells) derived from a single donor can offer safety advantages, such as limiting the exposure of the cells to only the complement of a single donor of viruses, microorganisms, or other potential pathogens, and thus minimizing the risk of disease transmission compared to the collection of cells from multiple donors. It is also formally possible for the iPSC to be derived from somatic cells of two or more donors in some embodiments. In some embodiments, the iPSC is derived from an adult skin fibroblast biopsy.

[0043] Embryonic stem cells are another type of pluripotent stem cells. In the methods, compositions, and kits of some embodiments herein, it is possible to use embryonic stem cells as pluripotent cells. Methods for isolating and preparing embryonic stem cells, including human embryonic stem cells, are described, for example, in U.S. Patent No. 6,200,806, which is hereby incorporated by reference in its entirety. However, it is also recognized that iPSCs may offer several advantages over embryonic stem cells in some embodiments. For example, ES cells may have constraints including ethical and legal regulations surrounding the destruction of the human embryo, which is one way to generate pluripotent cells. Chemically induced pluripotent cells are a preferred source.

[0044] Fibroblasts "Fibroblasts" are used herein according to their ordinary meaning in the art and include a class of cells that provide the structural framework (stroma) to various animal tissues. Fibroblasts can also migrate to the site of a wound to mediate wound healing and can become components of various connective tissues. Fibroblasts can be identified, for example, using fibroblast-specific antibodies, such as the antibody TE-7 described in Goodpaster et al., (2008), J. Histochem Cyotchem, 56: 347-58, which is hereby incorporated by reference in its entirety.

[0045] Without being limited to theory, any mesenchymal cell that adheres to a substrate (e.g., plastic) in cell culture, proliferates in the presence of serum, and is capable of synthesizing and depositing fibrillar collagenous ECM collagen can be used to make human ECM during culture. In some embodiments, fibroblasts, a type of mesenchymal cell, are used to produce the ECM. Exemplary types of cells that can produce ECM (sometimes referred to as "ECM-producing cells") include bone marrow mesenchymal stem cells (MSCs), iPSC-derived MSCs, ES-derived MSCs, and fibroblasts derived from, for example, neonatal foreskin fibroblast cell lines (which may be pluripotent or non-pluripotent), or skin or blood biopsies, but are not limited thereto. Without being limited to theory, it is further contemplated that the differences between mesenchymal cell types can be less than the differences between any cells or cell lines of a single donor strain (derived from a common cell source). If the cells are derived from a human donor sample containing connective tissue, these cells may be suitable for making ECM in cell culture according to some embodiments of the present specification.

[0046] Conventionally, fibroblasts were obtained from neonatal foreskins. The reason is that they can be easily obtained from discarded tissues. However, this approach generally requires cells from multiple donors to generate and screen a cell bank on a production scale. The use of more than one donor may also increase the risk of transmission of exogenous agents. The cost of developing and screening a donor cell bank can also be significant and disadvantageous. The inspection requirements for exogenous agents can also be another obstacle to banking and using cells from multiple donors. Furthermore, primary cell lines can be theoretically limited by the Hayflick limit for non-transformed cells, but in practice, primary cell lines may only be suitable for about 8 to 20 passages after isolation from the tissue source. These limitations regarding passage can limit the suitability of conventional fibroblasts for expansion along the process of a cell banking process, such as a master cell bank, a working cell bank, or a production cell bank scheme for manufacturing commercial quantities. However, the methods, kits, and compositions according to some embodiments herein can provide advantages over conventional sources of fibroblasts. For example, fibroblasts differentiated from iPSCs according to some embodiments herein can be from a single donor (e.g., if the iPSCs are expanded before differentiating into fibroblasts), reducing the risk of disease transmission and reducing inspection requirements. Furthermore, in some embodiments, iPSCs can undergo more expansion cycles than primary cell lines, facilitating the commercial scalability of fibroblasts or other ECM-producing cells derived from iPSCs. In some embodiments, iPSCs are expanded in the absence of serum.

[0047] In some embodiments, fibroblasts can be obtained by differentiating iPSCs into fibroblasts. Without being limited by theory, it is believed that iPSCs can be differentiated into fibroblasts, for example, by contacting the iPSCs with one or more fibroblast differentiation factors that may include, for example, one or more growth factors. As used herein, "fibroblast differentiation factor" (including variations of this base term) refers to a set of gene products and / or nucleic acids encoding gene products that are sufficient to differentiate pluripotent cells (e.g., iPSCs) into fibroblasts. For example, it has been reported that iPSCs can be differentiated into fibroblasts by contacting them with connective tissue growth factor (CTGF). iPSCs can be grown on a 3-D scaffold. Xu et al., Scientific Reports 5: 8480 DOI: 10.1038 / srep08480, which is hereby incorporated by reference in its entirety. Thus, in some embodiments, the fibroblast differentiation factor comprises, consists essentially of, or consists of CTGF. In some embodiments, the fibroblast differentiation factor comprises, consists essentially of, or consists of CTGF and fibrinogen. In some embodiments, the iPSCs are cultured on a three-dimensional substrate (e.g., dextrin microcarrier; see, e.g., U.S. Patent No. 6,378,527, which is hereby incorporated by reference in its entirety) and then contacted with the fibroblast differentiation factor.

[0048] As used herein, "producing fibroblast" refers to a fibroblast that is being used or can be used for the production of ECM by the methods, compositions, and kits of some embodiments herein.

[0049] As used herein, "precursor fibroblast" refers to a fibroblast that is being used or can be used as a precursor of pluripotent cells. For example, according to the methods and kits of some embodiments, the precursor fibroblast can be contacted with a dedifferentiation factor to dedifferentiate the precursor fibroblast into iPSCs.

[0050] Cell Culture and Substrate Various approaches to cell culture can be used with the methods, kits, and compositions of some embodiments herein. Detailed guidance regarding cell culture protocols and reagents can be found, for example, in Sambrook et al., "Molecular Cloning: A Laboratory Manual (3rd Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2000, which is hereby incorporated by reference in its entirety. Generally, the culture of fibroblasts for the production of ECM by the methods, compositions (compositons), and kits of some embodiments herein can be performed in a culture medium.

[0051] In some embodiments, the cell culture medium contains serum. The serum may initially be part of the cell culture medium or may be added later in the culture process. Suitable types of serum for use with the methods, compositions, and kits of some embodiments herein include tested clinical grade fetal bovine serum, pooled human serum from expired unit blood, or combinations of these two substances. In some embodiments, the amount (v / v) of serum in the culture medium is from about 0.1% to about 20%, such as from about 0.1% to about 15%, from about 0.1% to about 10%, from about 0.1% to about 5%, from about 0.1% to about 3%, from about 0.1% to about 1%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, from about 1% to about 5%, from about 1% to about 3%, from about 3% to about 20%, from about 3% to about 35%, from about 3% to about 30%, from about 3% to about 5%, from about 5% to about 20%, from about 5% to about 15%, from about 5% to about 10%, from about 10% to about 20%, or from about 15% to about 20%, such as about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (including ranges between any two of the recited values). In some embodiments, the amount (v / v) of serum in the culture medium is from about 0.1% to about 10%. In some embodiments, the amount of serum in the culture medium is an amount sufficient to increase the biological production of ECM by the cells in culture. In some embodiments, the amount of serum in the culture medium is an amount sufficient to induce the maturation and cross-linking of the ECM produced by the cells in culture. As used herein, "cross-linked" ECM is used herein according to its ordinary meaning in the art and includes ECM in which a polypeptide (e.g., collagen) is directly or indirectly bound to one or more other polypeptides of the ECM by ionic and / or covalent bonds other than peptide bonds. In some embodiments, each polypeptide of the cross-linked ECM is covalently and / or ionically bound by a bond other than a peptide bond to at least one other polypeptide of the cross-linked ECM. In some embodiments, the cross-linked peptide of the ECM is directly bound to another peptide of the ECM.In some embodiments, the cross-linked polypeptides of the ECM are indirectly bound to another polypeptide of the ECM, for example, via intervening small molecules, ions, amino acids, and / or different polypeptides. In some embodiments, the cross-linked ECM comprises an ECM in which most, substantially all, or all of the polypeptides, for example, about or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (including ranges between any two of the recited values) of the polypeptides are bound to at least one other polypeptide of the ECM by non-peptide bonds.

[0052] In some embodiments, pluripotent cells (e.g., iPSCs) or fibroblasts are cultured on or near a substrate. In some embodiments, the substrate comprises, consists of, or consists essentially of dextran, such as a dextran microcarrier. Advantageously, the dextran substrate can then be digested using dextranase as described herein, which can facilitate the isolation and purification of the ECM produced by cell culture according to some embodiments herein. In some embodiments, the substrate comprises, consists of, or consists essentially of a carbohydrate. In some embodiments, the substrate comprises, consists of, or consists essentially of a polymer. In some embodiments, the substrate comprises, consists of, or consists essentially of plastic. In some embodiments, the step of isolating the ECM comprises separating the ECM from any substrate or solid phase. In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). Without being bound by theory, it is believed that dextranase does not act as a protease on the ECM, and thus the step of isolating the ECM using dextranase according to some embodiments herein results in an intact (non-protease digested ECM). In some embodiments, the isolated ECM has not been digested by any protease and is thus intact.

[0053] Dextran is a large branched carbohydrate made up of many glucose molecules. The dextran chains may be of various lengths and may have, for example, a molecular weight ranging from as little as 3 kDa to over 2000 kDa. Dextran structures such as dextran microcarriers are thought to be useful as substrates for the culture of pluripotent cells or cells such as fibroblasts in the methods, kits, and compositions of some embodiments herein. Advantageously, dextran can be non-toxic and can be readily hydrolyzed by dextranase as described in more detail herein.

[0054] Dextranase is a bacterial enzyme widely used in industrial applications (EC 3.2.1.11, dextran hydrolase, endodextranase, dextranase DL 2, DL 2, endodextranase, alpha-D-1,6-glucan-6-glucanohydrolase, 1,6-alpha-D-glucan 6-glucanohydrolase), and has the tissue name 6-alpha-D-glucan 6-glucanohydrolase. This enzyme catalyzes the following chemical reaction, namely the endohydrolysis of the (1->6)-alpha-D-glucoside bond of dextran. In cell culture, dextranase can be used for various purposes, for example, for the production of cell products and cell-derived virus products or other biological agents using dextran microcarriers (see, for example, U.S. Patent No. 6,378,527), or for the disposal of large amounts of bead waste used in commercial-scale cell culture for the production of biological products, and can be used for the isolation of expanded mammalian cells and mammalian cell lines such as human chondrocytes. In some embodiments of the methods, kits, and compositions, dextran can be used as a substrate or scaffold for pluripotent cells (e.g., iPSCs), or fibroblasts derived from pluripotent cells (e.g., fibroblasts produced by dedifferentiating fibroblasts into iPSCs and then redifferentiating the iPSCs into fibroblasts). In some embodiments, dextran is not used for the culture and / or expansion of chondrocytes. In some embodiments, dextran is not used for the culture of non-human cells, such as VERO (monkey) and / or CHO (rodent) cells.

[0055] The use of dextranase enzymes in various research-grade small-scale cultures that are generally not applicable to commercial use or human ECM has been disclosed by Pinney et al. (see Pinney E, (2011) International Journal of Stem Cells 4: 70-75; and Menen et al., (2012) Anticancer Research 32: 1573-1578. Each is hereby incorporated by reference in its entirety). It is noted that conventional approaches required the use of dextran for cell isolation after expansion, mainly in the context of waste treatment of dextran microcarriers. In some embodiments, dextran is used in the culture of pluripotent cells or fibroblasts that produce ECM. In some embodiments, dextran is used in the culture of pluripotent cells or fibroblasts but not for waste treatment. In some embodiments, the culture contains human ECM produced by pluripotent cells or fibroblasts.

[0056] In the methods, compositions, and / or kits of some embodiments, the ECM is produced by cells in culture (e.g., fibroblasts and / or iPSCs), and then the ECM is isolated from the cells in culture. In some embodiments, the step of isolating the ECM includes separating the ECM from a substrate or solid phase. In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). In some embodiments, the isolated ECM has not been digested by any protease and is thus intact. In some embodiments, the step of isolating the ECM includes isolating the soluble fraction of the ECM. The spent medium containing the soluble ECM may be isolated from the cell culture and optionally replaced with fresh medium to allow the cell culture to continue to produce. In some embodiments, the ECM is isolated by acid washing at a pH that promotes the enzymatic activity of dextranase (e.g., pH 6.0 - 6.5) followed by dextranase digestion. The dextranase may be at a relatively low concentration (less than 1000 U / ml, e.g., 1000 U / ml, 900 U / ml, 800 U / ml, 700 U / ml, 600 U / ml, 500 U / ml, 400 U / ml, 300 U / ml, 200 U / ml, 100 U / ml, 50 U / ml, 10 U / ml, 5 U / ml, 2 U / ml, or less than 1 U / ml (including ranges between any two of these values, e.g., 1 - 1000 U / ml; 500 - 1000 U / ml, 1 - 500 U / ml, 100 - 300 U / ml, or 600 - 800 U / ml)). The lower concentration of dextranase is thought to have a lower likelihood of non-specifically degrading other non-dextran carbohydrates, some of which may be components of the ECM. The ECM may be further contacted with DNase to remove cellular nucleic acids. The production of the ECM in some embodiments may thus be without the use of animal or plant proteins using xeno-free culture media and without the use of Pinney to regulate ex vivo cell expansion.

[0057] Production of ECM using fibroblasts generated from pluripotent cells In some embodiments, a method of manufacturing an extracellular matrix is provided. The method may include differentiating pluripotent cells (such as induced pluripotent stem cells (iPSCs)) into producer fibroblasts. The method may include culturing the producer fibroblasts such that the producer fibroblasts produce an ECM. The method may include isolating the ECM from the producer fibroblasts and thus manufacturing the ECM. In some embodiments, the method includes dedifferentiating progenitor fibroblasts to form pluripotent cells before differentiating the pluripotent cells into producer fibroblasts. In some embodiments, the pluripotent cells are expanded before being differentiated into fibroblasts. It is noted that by first expanding the pluripotent cells, a large population of pluripotent cells can be generated from a small number of primordial pluripotent cells before redifferentiating the pluripotent cells into fibroblasts. In some embodiments, the pluripotent cells are expanded at least about 10-fold, such as about or at least about 10, 15, 20, 25, or 30-fold (including ranges between any two of the recited values, such as about 10-30 fold, about 10-25 fold, about 10-20 fold, about 15-30 fold, about 15-25 fold, about 15-20 fold, or about 20-30 fold). In some embodiments, the pluripotent cells are expanded at least about 15-fold. It is noted that the ability of pluripotent cells to undergo multiple doublings provides an advantage over conventional sources of fibroblasts, such as neonatal foreskin cells, which typically undergo only about 10-12 doublings before exhausting and senescing.

[0058] Figure 1 is a flow diagram illustrating a method of manufacturing an ECM using fibroblasts differentiated from iPSCs, according to some embodiments of the present specification. In the method, optionally, progenitor fibroblasts may be dedifferentiated to form iPSCs 100. In the method, induced pluripotent stem cells (iPSCs) may be differentiated into producer fibroblasts 110. In the method, the producer fibroblasts may be cultured, whereby the producer fibroblasts produce an extracellular matrix (ECM) 120. In the method, the producer fibroblasts may be cultured, whereby the producer fibroblasts produce an extracellular matrix (ECM) 130.

[0059] In the methods, kits, and compositions of some embodiments, precursor fibroblasts (or iPSCs) from a single donor are used to create the ECM. Advantageously, the process of manufacturing ECM from a single donor can minimize the number of potential contaminants (such as viral contaminants). Thus, in some embodiments, the iPSCs that differentiate into producing fibroblasts are derived from only a single donor. It is also noted that virus-free (e.g., retrovirus-free) pluripotent cells can further minimize the risk of contamination or immunogenicity. Thus, in some embodiments, the iPSCs are free of viral insertions encoding dedifferentiation factors such as the Oct family members, Sox family members, and Klf family members described herein. In some embodiments, the method does not include the step of using any of embryonic stem (ES) cells, bone marrow multipotent stem cells (MSCs), ES-derived MSCs, or non-pluripotent neonatal foreskin fibroblast cell lines. In some embodiments, the method does not include the step of using any of embryonic stem (ES) cells, bone marrow multipotent stem cells (MSCs), ES-derived MSCs, or any neonatal foreskin fibroblast cell lines. In some embodiments, the iPSCs are footprint-free (e.g., iPSCs dedifferentiated using chemical dedifferentiation factors or mRNA dedifferentiation factors).

[0060] It is noted that pluripotent cells can be differentiated into producing fibroblasts using the differentiation factors described herein. In some embodiments, the pluripotent cells are expanded before differentiating the cells into producing fibroblasts. In some embodiments, the step of expanding the pluripotent cells before differentiation can provide a large number of cells from a single donor, which can result in a large number of producing fibroblasts from a single donor upon differentiation. In some embodiments, after expansion, but before differentiation into producing fibroblasts, the pluripotent cells are banked. For example, the pluripotent cells may be banked by freezing in liquid nitrogen.

[0061] In some embodiments, the producing fibroblasts are cultured in a medium consisting essentially of, or consisting of, terminally differentiated cells (e.g., the producing fibroblasts themselves). In some embodiments, the producing fibroblasts are cultured in a medium substantially free of, or free of, mesenchymal stem cells (MSCs). Without being limited by theory, it is believed that fibroblasts differentiated from fibroblast-derived iPSCs by the methods, compositions, and kits of some embodiments herein may be effective for the efficient large-scale production of mature ECM. For example, the process of dedifferentiating, expanding, and redifferentiating a single donor iPSC according to some embodiments can result in commercially scaled ECM-producing fibroblasts with minimal risk of contamination or disease transmission.

[0062] In some embodiments, the method is performed in the absence of any of embryonic stem (ES) cells, bone marrow multipotent stem cells (MSCs), ES-derived MSCs, non-pluripotent neonatal foreskin fibroblast cell lines, or any neonatal foreskin fibroblast lines, or two or more of these. In some embodiments, the method is performed in the absence of any of embryonic stem (ES) cells, bone marrow multipotent stem cells (MSCs), ES-derived MSCs, or non-pluripotent neonatal foreskin fibroblast cell lines, or two or more of these. In some embodiments, the method is performed in the absence of any of these. In some embodiments, the method is performed on a commercial scale, thus achieving commercial scale production of ECM. In some embodiments, the method includes commercial scale production that includes culturing at least 10 liters of a fibroblast culture, such as at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 liters of a fibroblast culture (including ranges between any two of the recited values, such as 10 - 1000 liters, 10 - 500 liters, 10 - 200 liters, 50 - 1000 liters, 50 - 500 liters, 50 - 200 liters, 100 - 1000 liters, 100 - 500 liters, or 100 - 200 liters). In some embodiments, the method includes commercial scale production that includes production of at least 50 g of ECM, such as at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 g of ECM (including ranges between any two of the recited values). As noted above, primary cells for commercial scaling, such as primary bone marrow MSCs, or cells from neonatal foreskin, may require cells from multiple donors, which can increase the risk of contamination or disease transmission. Also, as noted above, ES cells can increase the risk of disease transmission and may be subject to availability and use restrictions, for example due to ethical regulations. Further, MSCs such as bone marrow MSCs may remain multipotent and thus be subject to further differentiation.On the one hand, the methods, compositions, and kits in some embodiments can advantageously be carried out using differentiated fibroblasts (but not MSCs) such that the fibroblasts do not undergo further differentiation.

[0063] In some embodiments, the method includes the step of dedifferentiating a progenitor fibroblast to form an iPSC. The progenitor fibroblast can be contacted with a dedifferentiation factor, thereby dedifferentiating the progenitor fibroblast into an iPSC. In some embodiments, the progenitor fibroblast includes adult dermal (biopsy) fibroblasts. In some embodiments, the progenitor fibroblast is from a single donor. In some embodiments, the iPSC has no viral insert encoding an Oct family member, a Sox family member, or a Klf family member. In some embodiments, the iPSC is footprint-free. The iPSC can then be expanded and differentiated into a production fibroblast.

[0064] The step of culturing the producing fibroblasts may be performed in the presence of oxygen. Oxygen is involved in all or essentially all metabolic processes to promote efficient and maximal conversion of carbon sources, and without being limited by theory, culturing in normoxia is considered advantageous. On a commercial scale, oxygen is a reactant in the reaction that converts glucose and glutamine to ATP, which in turn supports the synthesis of, among other proteins, collagen in particular. Under hypoxia, cells have no choice but to rely much more on glycolysis rather than aerobic respiration, which leads to inefficient synthesis of proteins such as collagen. Therefore, in some embodiments, the step of culturing the producing fibroblasts is performed under normoxia. In some embodiments, the step of culturing the producing fibroblasts is performed under hypoxia. Hypoxia generally refers to an oxygen concentration lower than the oxygen concentration in ambient air (normoxia; about 15% - 20% oxygen). In some embodiments, the hypoxic conditions include an oxygen concentration of less than about 10%. In some embodiments, the hypoxic conditions are characterized by an oxygen concentration of about 1% - 10%, 1% - 9%, 1% - 8%, 1% - 7%, 1% - 6%, 1% - 5%, 1% - 4%, 1% - 3%, or 1% - 2%. The hypoxic conditions may be created and maintained using a culture apparatus capable of controlling the ambient gas concentration, such as an anaerobic chamber.

[0065] In some embodiments, the producing fibroblasts are cultured in the presence of a substrate. In some embodiments, the substrate includes a polymer, such as a plastic surface or dextran. In some embodiments, the substrate includes a dextran microcarrier.

[0066] For example, in some embodiments where the producing fibroblasts are cultured in the presence of dextran microcarriers, the step of isolating the ECM from the producing fibroblasts includes washing the ECM in an acidic buffer and contacting a solution containing the producing fibroblasts and the ECM with dextranase. The solution containing the producing fibroblasts and the ECM may also be contacted with DNase. By way of example, the dextranase may include bacterial dextranase. In some embodiments, the dextranase is provided at a concentration of 1 to 1000 U / ml. The dextranase and DNase are thus thought to be able to promote the purification of the ECM by removing other substances. Accordingly, the step of isolating the ECM may include separating the ECM from a substrate or solid phase. In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). In some embodiments, the isolated ECM is not digested by any protease and is therefore intact. In some embodiments, the step of purifying human ECM includes washing with an acidic buffer at pH 6.0 to 6.5 to remove residual culture medium components. The acid-washed ECM may be contacted with a solution containing 1 to 1000 U / ml of bacterial dextranase and 1 to 1000 U / ml of recombinant human DNse in an acidic solution having a pH of 2.0 to 7.0, or preferably pH 6.0 to 6.5, where the enzyme activity is sufficient to remove dextran beads and cellular nucleic acids. Optionally, the purification may be performed after in-process inspection of the ECM.

[0067] In some embodiments, when isolated from the producing fibroblasts, the ECM is no longer in fluid communication with the producing fibroblasts. For example, the ECM can be in a separate container from the producing fibroblasts. In some embodiments, the step of isolating the ECM from the producing fibroblasts comprises purifying the ECM to produce a composition that is at least 20% (w / w) ECM, such as about or at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (including ranges between any two of the recited values) ECM. In some embodiments, the composition comprises at least about 80% ECM. In some embodiments, the composition comprises at least about 80% - 95% ECM. In some embodiments, when isolated from the producing fibroblasts, the ECM is no longer bound to the substrate (e.g., no longer directly bound to the substrate and / or no longer bound to molecules that are directly or indirectly bound to the substrate via one or more intervening binding molecules).

[0068] The ECM may include the mature ECM components described herein. "Mature ECM" is used herein according to its ordinary meaning in the art and is a cross-linked ECM that includes an ECM that includes the c-terminal propeptide of COL1, triple helix or non-reduced gamma-type fibrillar collagen, or a combination of two or more of these features. In some embodiments, the mature ECM includes triple helix and / or non-reduced gamma-type fibrillar collagen. In some embodiments, the mature ECM includes collagen. In some embodiments, about 90% (w / w) of the ECM includes COL1, and about 10% is selected from the group consisting of COL3, COL4, COL5, COL6, or any combination or all of these. Exemplary sequences of Homo sapiens COL1, COL3, COL4, COL5, and COL6 include, but are not limited to, the sequences shown in Table 1 herein. In some embodiments, at least about 80% of the mature ECM includes COL1, and at least about 10% is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof. In some embodiments, at least about 85% of the mature ECM includes COL1 (e.g., at least about 85%, 87%, 90%, or 95%), and at least about 5% (e.g., at least about 5%, 10%, 13%, or 15%) is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof. In some embodiments, the mature ECM includes the c-terminal propeptide of COL1, or triple helix or non-reduced gamma-type fibrillar collagen, or both. In some embodiments, the mature ECM includes triple helix and / or non-reduced gamma-type fibrillar collagen. It is noted that different molecules of mature ECM, such as the collagen molecules described herein, can be readily detected using, among other assays, ELISA in particular. In some embodiments, antibodies specific for the collagen protein (or the c-terminal propeptide of COL1) are used in quantitative ELISA to ascertain the amounts of the components of the mature ECM.

[0069] In some embodiments, the method further comprises contacting the producing fibroblasts with serum until the producing fibroblasts produce mature collagen. Without being limited by theory, it is believed that contacting the producing fibroblasts with serum may support ECM precipitation, increase biological production, and induce the production of mature ECM, such as cross-linked ECM. Then, the amount of serum can be gradually reduced until there is at least a 95% reduction in serum concentration. In some embodiments, the gradual reduction occurs over at least about 5 days, such as about or at least about 5, 7, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 days (including the range between any two of the recited values).

[0070] In some embodiments, to initiate a production lot of fibroblasts, vials of pluripotent cells (e.g., iPSCs generated by contacting fibroblasts with dedifferentiation factors) are expanded to an appropriate number, and the pluripotent cells are then induced to dedifferentiate into fibrotic collagenous ECM-producing cells such as MSCs or fibroblasts, and these cells are utilized to produce mature ECM. Thus, in some embodiments, a method for manufacturing ECM comprises (a) differentiating a fibroblast skin biopsy or blood sample, (b) subsequently inducing pluripotency via a chemical polypeptide, or nucleic acid-based vector-free / footprint-free induced pluripotency, (c) isolating clones and expanding cells in a pluripotent state, (d) generating a cell bank (optionally including characterization of the cells and testing for exogenous agents), (e) expanding pluripotent cells to initiate a production lot of ECM, and / or (f) redifferentiating pluripotent cells into a differentiated state that produces sufficient mature fibrotic collagenous ECM that is insoluble under culture conditions.

[0071] In some embodiments, a kit for manufacturing an ECM is provided. The kit may include a composition containing human fibroblasts, a dedifferentiation factor, and a fibroblast differentiation factor. In some embodiments, the composition containing human fibroblasts includes cryopreserved human fibroblasts. In some embodiments, all of the fibroblasts in the composition are from a single donor. In some embodiments, the kit further includes a substrate, such as a dextran microcarrier. In some embodiments, the kit further includes dextranase and DNAase, which may be useful, for example, in isolating the ECM from cells and cell cultures. In some embodiments, the kit includes pluripotent cells, such as iPSCs, and a dedifferentiation factor instead of human fibroblasts (however, still includes a differentiation factor for differentiating the pluripotent cells into fibroblasts). In some embodiments, the kit does not contain protease.

[0072] Some embodiments include a composition comprising at least about 80% (w / w) extracellular matrix, wherein the extracellular matrix is produced according to any one of the methods described above. In some embodiments, the composition comprises about or at least about 60% (w / w) ECM, such as at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (including the range between any two of the recited values) of ECM. In some embodiments, the ECM of the composition consists essentially of, consists of, or includes the mature ECM described herein.

[0073] In some embodiments, the cell culture comprises a plurality of iPSC-derived fibroblasts that produce an extracellular matrix. The cell culture may further comprise a dedifferentiation factor described herein. In some embodiments, at least about 5% of the composition, such as at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% (w / w) (including ranges between any two of the recited values) is ECM. In some embodiments, about 90% (w / w) of the ECM comprises COL1, and at least about 10% is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof. In some embodiments, at least about 80% of the ECM comprises COL1, and at least about 10% is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof. In some embodiments, at least about 85% of the ECM comprises COL1 (e.g., at least about 85%, 87%, 90%, or 95%), and at least about 5% (e.g., at least about 5%, 10%, 13%, or 15%) is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof. In some embodiments, the ECM comprises the C-terminal propeptide of COL1, or triple helix or non-reducing gamma-type fibrillar collagen, or both. In some embodiments, the ECM comprises triple helix and / or non-reducing gamma-type fibrillar collagen.

[0074] Production of mature xeno-free ECM Some embodiments include a method of manufacturing an extracellular matrix (ECM). The method may include culturing fibroblasts and / or mesenchymal stem cells (MSCs) on a substrate. The substrate may include at least two surfaces. The culturing may be performed serum-free and xenofree until the fibroblasts and / or MSCs define a three-dimensional shape on at least two surfaces and until at least 80% (e.g., at least 80%, 85%, 90%, 93%, 95%, 97%, or 99%) of the fibroblasts and / or MSCs arrest their cell cycle. The fibroblasts and / or MSCs may then be contacted with serum for about or at least about two weeks (e.g., at least about 2, 3, 4, 5, 6, 7, or 8 weeks). As a result of the contact with serum, the fibroblasts and / or MSCs can produce a soluble mature ECM, and in this way, a solution containing the soluble mature ECM as well as the fibroblasts and / or MSCs can be produced. The solution may be xenofree. The method may further include isolating the soluble mature ECM from the fibroblasts and / or MSCs, and thus manufacturing an ECM that is a mature xenofree ECM. For example, the step of isolating the soluble mature ECM may include collecting the soluble fraction of the solution containing the spent medium. Also, in some embodiments, it is contemplated that the method may be performed such that (instead of adding serum later) the fibroblasts and / or MSCs are in a medium containing serum at the start of the culturing. Although fibroblasts and ECM have been described above, it is noted that it is clearly contemplated that the method may also be performed using the ECM-producing cells described herein. In some embodiments, the step of isolating the ECM includes separating the ECM from a substrate or solid phase. In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). In some embodiments, the isolated ECM is not digested by any protease and is thus intact.

[0075] Figure 2 is a flow diagram illustrating a method of manufacturing an ECM that includes culturing fibroblasts or MSCs for at least two weeks according to some embodiments of the present specification. In the method, optionally, a human pluripotent cell culture may be expanded. The expansion may be serum-free and xenofree, thereby producing human pluripotent cells. The human pluripotent cells may be contacted with a differentiation factor, whereby the human pluripotent cells differentiate into fibroblasts or MSCs. 200. In the method, fibroblasts and / or mesenchymal stem cells (MSCs) may be cultured on a substrate. The substrate may include at least two surfaces. The culture may be serum-free and xenofree, such that the fibroblasts and / or MSCs define a three-dimensional shape on at least two surfaces and the culture is carried out until at least 80% of the fibroblasts and / or MSCs arrest their cell cycle. 210. In the method, the fibroblasts and / or MSCs may be contacted with serum for at least about two weeks, thereby producing a soluble mature ECM by the fibroblasts and / or MSCs, and thereby producing a solution containing the soluble mature ECM and the fibroblasts or MSCs, the solution being xenofree. 220. In the method, the soluble mature ECM may be isolated from the producing fibroblasts, thereby producing an ECM that is a mature xenofree ECM. 230.

[0076] In some embodiments, the method further includes expanding a human pluripotent cell culture (e.g., iPSCs such as footprint-free iPSCs described herein). The expansion may be carried out under serum-free and xenofree conditions, and an expanded amount of human pluripotent cells can be produced. The method may further include contacting the expanded human pluripotent cells with a differentiation factor described herein. The differentiation factor may be suitable for differentiating the human pluripotent cells into fibroblasts or MSCs.

[0077] It has been observed herein that culturing fibroblasts and / or MSCs with serum for at least two weeks or more unexpectedly results in the production of a mature ECM containing cross-linked and highly soluble collagen. Thus, in some embodiments, the contact of fibroblasts and / or MSCs with serum is carried out for at least about two weeks, e.g., at least about two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks (including the range between any two of the recited values). In some embodiments, the contact of fibroblasts and / or MSCs with serum is carried out for at least about eight weeks. In some embodiments, the contact of fibroblasts and / or MSCs with serum is carried out for about two weeks to about twelve weeks, e.g., about two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks (including the range between any two of the recited values, e.g., about two weeks to about eleven weeks, about two weeks to about ten weeks, about two weeks to about eight weeks, about two weeks to about six weeks, about two weeks to about four weeks, about three weeks to about twelve weeks, about three weeks to about ten weeks, about three weeks to about eight weeks, about three weeks to about six weeks, about four weeks to about twelve weeks, about four weeks to about ten weeks, about four weeks to about eight weeks, about four weeks to about six weeks, about six weeks to about twelve weeks, about six weeks to about ten weeks, or about six weeks to about eight weeks). In some embodiments, the contact is carried out for about two weeks to about eight weeks. In some embodiments, the contact is carried out for longer than two weeks. In some embodiments, the fibroblasts and / or MSCs are cultured with serum under normoxic conditions. In some embodiments, the fibroblasts and / or MSCs are cultured with serum under hypoxic conditions.

[0078] In some embodiments, the human pluripotent cells include induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are footprint-free. In some embodiments, the iPSCs are derived from a single donor. As discussed herein, the use of iPSCs allows for the expansion and optionally banking of pluripotent cells prior to differentiating them into ECM-producing cells such as fibroblasts with the methods, compositions, and kits of some embodiments. Thus, since the iPSCs are derived from a single donor and can then be expanded, such use of iPSCs can enable the production of ECM-producing cells from a single donor in commercial-scale amounts.

[0079] In some embodiments, the method further includes the step of manufacturing a cosmetic composition comprising mature xeno-free ECM. In some embodiments, the spent medium may also be useful in the manufacture of some cosmetic compositions.

[0080] When serum is used or added to cultures of ECM-producing cells such as fibroblasts with the methods, compositions, and kits described herein, ascorbic acid may also be useful, for example, in maintaining the health of the cells during culture. It is noted that, without being limited by theory, ascorbic acid may be required for certain post-transcriptional modifications of collagen that stabilize mature triple-helical collagen. By way of example, ascorbic acid can serve as a cofactor for some enzymes such as prolyl hydroxylase. Thus, in some embodiments, the method further includes the step of contacting the fibroblasts or MSCs with ascorbic acid while the cells are in contact with serum (e.g., for at least two weeks).

[0081] Without being limited to theory, it is believed that cells such as fibroblasts and / or MSCs can support ECM precipitation and maturation when they reach a sufficient density on a substrate (such as a scaffold or support, e.g., the dextran microcarriers described herein). Further, when the cells begin to reach a sufficient density, their cell cycle can arrest (i.e., the cells can enter the G0 phase of the cell cycle). Thus, in some embodiments, fibroblasts or MSCs are not contacted with serum until they reach an appropriate density to support ECM precipitation and maturation. In some embodiments, fibroblasts or MSCs are not contacted with serum until these fibroblasts or MSCs precipitate on at least two surfaces of a substrate defining a three-dimensional shape and at least about 70% of the fibroblasts or MSCs arrest their cell cycle. In some embodiments, fibroblasts or MSCs are not contacted with serum until these fibroblasts or MSCs precipitate on at least two surfaces of a substrate defining a three-dimensional shape and at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (including the range between any two of the recited values) of the fibroblasts or MSCs arrest their cell cycle.

[0082] Some suitable forms of serum, such as tested clinical-grade fetal bovine serum or pooled human serum from expired unit blood, or combinations thereof, can be contacted with fibroblasts and / or MSCs. In some embodiments, the step of contacting serum with fibroblasts includes adding a solution containing fibroblasts and / or MSCs to the serum. "Adding" is used broadly herein and includes adding serum to a solution containing fibroblasts and / or MSCs as well as adding a solution containing fibroblasts and / or MSCs to the serum. In some embodiments, serum is added to a solution containing fibroblasts and / or MSCs until the volume / volume (v / v) amount of serum is about 0.1% to 10%, such as about 1 to 2%, 1 to 5%, 2 to 10%, 2 to 5%, 3 to 10%, 3 to 5%, or 5 to 10%.

[0083] Advantageously, the process of producing xeno-free ECM according to some embodiments herein can reduce the risk of harmful immune responses in users of products containing the ECM. Such products can further enjoy advantages such as simplification of regulatory authority review. In some embodiments, the fibroblasts and / or MSCs are derived from human pluripotent cells (e.g., iPSCs) of cell lines previously expanded using animal components. In some embodiments, the pluripotent cells are from a single donor. The pluripotent cells can be expanded without using xenogeneic components. In this way, a large amount of xeno-free cells can be obtained. The xeno-free pluripotent cells according to some embodiments can then be differentiated into ECM-producing cells, such as fibroblasts and / or MSCs. Differentiation may include contacting the xeno-free pluripotent cells with the differentiation factors described herein.

[0084] Furthermore, the xeno-free ECM produced by the methods, compositions, and kits of some embodiments can include the mature ECM described herein. Therefore, the mature xeno-free ECM can be well-suited for some cosmetics and medical products. In some embodiments, the mature xeno-free ECM includes fibrillar collagen. In some embodiments, the mature xeno-free ECM includes the C-terminal propeptide of COL1, or triple helix or non-reduced gamma-type fibrillar collagen, or both. In some embodiments, the mature xeno-free ECM includes triple helix and / or non-reduced gamma-type fibrillar collagen.

[0085] In some embodiments, the solution has at least about 100 μg of collagen per 1 cm 2 of substrate, e.g., at least about 100 μg of collagen per 1 cm 2 of substrate, at least about 100 μg of collagen per 1 cm 2 of substrate, 150 μg of collagen per 1 cm 2 of substrate, 200 μg of collagen per 1 cm 2 of substrate, 250 μg of collagen per 1 cm 2 of substrate, 300 μg of collagen per 1 cm 2 of substrate, 350 μg of collagen per 1 cm2 400 μg of collagen per 1 cm 2 450 μg of collagen per 1 cm 2 500 μg of collagen per 1 cm 2 600 μg of collagen per 1 cm 2 700 μg of collagen per 1 cm 2 800 μg of collagen per 1 cm 2 900 μg of collagen per 1 cm, or 1 cm 2 Contains 1000 μg of collagen per 1 cm (including the range between any two of the listed values). In some embodiments, the solution contains at least 250 μg of collagen per 1 cm2 of substrate.

[0086] In some embodiments, the produced mature xenogeneic-free ECM contains at least about 100 μg of collagen per 1 cm2 of substrate, for example 1 cm 2 At least about 100 μg of collagen per 1 cm 2 150 μg of collagen per 1 cm 2 200 μg of collagen per 1 cm 2 250 μg of collagen per 1 cm 2 300 μg of collagen per 1 cm 2 350 μg of collagen per 1 cm 2 400 μg of collagen per 1 cm 2 450 μg of collagen per 1 cm 2 500 μg of collagen per 1 cm 2 600 μg of collagen per 1 cm 2 700 μg of collagen per 1 cm 2 800 μg of collagen per 1 cm 2 900 μg of collagen per 1 cm, or 1 cm 2 Contains 1000 μg of collagen per 1 cm (including the range between any two of the listed values). In some embodiments, the produced mature xenogeneic-free ECM contains at least 250 μg of collagen per 1 cm2 of substrate.

[0087] In some embodiments, the method includes detecting the amount of mature ECM in a solution. In some embodiments, the detection is performed using ELISA to detect the presence, absence, and / or level of one or more components of the mature ECM described herein.

[0088] Some embodiments include a solution comprising fibroblasts or MSCs produced according to any one of the above methods and a soluble mature ECM. The solution may be xenofree, and the soluble mature ECM may include crosslinked collagen. In some embodiments, the solution includes fibroblasts but does not include MSCs. In some embodiments, the fibroblasts or MSCs are on a substrate in the solution, and the solution includes at least about 100 μg of collagen per cm2 of substrate, e.g., 1 cm 2 at least about 100 μg of collagen per cm, 1 cm 2 150 μg of collagen per cm, 1 cm 2 200 μg of collagen per cm, 1 cm 2 250 μg of collagen per cm, 1 cm 2 300 μg of collagen per cm, 1 cm 2 350 μg of collagen per cm, 1 cm 2 400 μg of collagen per cm, 1 cm 2 450 μg of collagen per cm, 1 cm 2 500 μg of collagen per cm, 1 cm 2 600 μg of collagen per cm, 1 cm 2 700 μg of collagen per cm, 1 cm 2 800 μg of collagen per cm, 1 cm 2 900 μg of collagen per cm, or 1 cm 2 1000 μg of collagen (including the range between any two of the listed values) per cm2 of substrate. In some embodiments, the solution includes at least 250 μg of collagen per cm2 of substrate.

[0089] The process of producing the described period ECM according to some embodiments of this specification can produce a larger amount of mature ECM than conventional methods. For example, the process of culturing cells for about 8 to 12 weeks before collecting the used medium containing soluble ECM according to some embodiments of this specification can produce more ECM than conventional methods. On the other hand, the production of embryonic-like ECM under hypoxic conditions by some conventional methods may result in a generally smaller amount of ECM that does not necessarily contain any mature ECM. The amount of ECM produced by a particular method can be measured, for example, by an SDS-PAGE gel that compares the ECM produced by a target cell culture with type I collagen from adult tissue.

[0090] Manufacture of ECM including gradual removal of serum Some embodiments include a method of manufacturing an extracellular matrix (ECM). The method may include providing fibroblasts in a medium containing a non-zero concentration of serum. The method may include gradually reducing the amount of serum in the medium containing fibroblasts until the medium contains a serum concentration of 5% or less, such as 5% or less, 4%, 3%, 2%, or 1%. This gradual reduction of serum may also be referred to herein as "serum withdrawal". After the gradual reduction of serum, the method may include culturing the fibroblasts for at least about one week (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, including the range between any two of the recited values), whereby the fibroblasts produce a soluble ECM. Thus, the method can produce a solution containing fibroblasts and soluble ECM. The method may further include isolating the soluble ECM from the fibroblasts and thus manufacturing the ECM. In some embodiments, the method includes little or no cell expansion once serum withdrawal begins. In some embodiments, the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is at least about 0.7 times the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. In some embodiments, the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is at least about 0.6 times, 0.7 times, 0.8 times, 0.9 times, 1 time, 1.1 times, or 1.2 times (including the range between any two of the recited values) the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. In some embodiments, the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is at least about 0.9 times the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. In some embodiments, the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is about 0.8 to 1.2 times the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. In some embodiments, the step of gradually reducing the amount of serum (i.e., serum withdrawal) is performed without cell expansion or cell subculture. In some embodiments, the step of gradually reducing the amount of serum (i.e., serum withdrawal) is performed without cell subculture. In some embodiments Then, the serum is completely removed by gradual reduction of the serum (except for trace amounts that do not significantly affect the culture or ECM). Although fibroblasts are described above, it is noted that the method can clearly also be carried out using the ECM-producing cells described herein. In some embodiments, the step of isolating the ECM from the fibroblasts includes separating the ECM from a substrate or solid phase (e.g., the substrate or solid phase may be directly or indirectly bound to the fibroblasts and / or the ECM may be bound to the substrate or solid phase during production by the fibroblasts). In some embodiments, the isolated ECM is separated from any substrate or solid phase (e.g., not bound to any substrate or solid phase). In some embodiments, the isolated ECM is not digested by any protease and is thus intact.

[0091] Figure 3 is a flow diagram illustrating a method of manufacturing an ECM, including serum withdrawal, according to some embodiments herein. The ECM may be xenofree. In the method, fibroblasts can be provided in a medium containing a certain concentration of serum. 300. In the method, the amount of serum in the medium containing fibroblasts can be gradually reduced until the medium contains a serum concentration of 5% or less. 310. In the method, the fibroblasts can be cultured for at least about two weeks (e.g., at least about 2, 3, 4, 5, 6, 7, or 8 weeks), whereby the fibroblasts produce soluble ECM, thereby producing a solution containing fibroblasts and soluble ECM. 320. In the method, the soluble ECM is isolated from the fibroblasts, thereby producing the ECM. 330.

[0092] As used herein, the step of gradually reducing the amount of serum in the medium can occur over several days, such as at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days (ranges between any two of the recited values, such as about 1 - 30 days, 1 - 20 days, 1 - 14 days, 1 - 10 days, 1 - 7 days, 1 - 5 days, 1 - 30 days, 2 - 20 days, 2 - 14 days, 2 - 10 days, 2 - 7 days, 2 - 5 days, 3 - 20 days, 3 - 14 days, 3 - 10 days, 3 - 7 days, 3 - 5 days, 5 - 20 days, 5 - 14 days, 5 - 10 days, 5 - 7 days, 7 - 20 days, 7 - 14 days, 7 - 10 days, 10 - 20 days, or 10 - 14 days). In some embodiments, the serum is gradually reduced for at least about 5 days. The gradual reduction of serum can involve the removal of serum-containing medium and replacement with serum-free medium. By performing multiple exchanges of a portion of the serum-containing medium with serum-free medium, the serum can be gradually reduced until it is effectively eliminated (i.e., such that only a trace amount of serum remains that does not significantly affect cell culture or ECM), without substantially degrading the ECM or inducing apoptosis.

[0093] Surprisingly, as described by some embodiments herein, by gradually removing serum until the serum is completely (or nearly completely) removed, a majority of the ECM-producing cells are induced to arrest the cell cycle (i.e., enter the G0 phase), and soluble mature ECM can be produced without substantially degrading the ECM or inducing apoptosis. Thus, in some embodiments, after the gradual reduction of serum, soluble intact and mature ECM is produced.

[0094] In some embodiments, after the gradual reduction of serum, at least about 70% of the fibroblasts in the solution, such as at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (including the range between any two of the recited values, such as 70-99%, 70-95%, 70-90%, 80-99%, 80-95%, 80-90%, 85-99%, 85-95%, or 85-90%) are in the G0 cell cycle stage. In some embodiments, at least about 90% of the fibroblasts in the solution are in the G0 cell cycle stage.

[0095] In some embodiments, after the gradual reduction of serum, less than about 5% of the fibroblasts in the solution are undergoing apoptosis. As used herein, cells “undergoing” apoptosis at a certain rate refer to cells that exhibit a detectable marker indicative of apoptosis, and thus may include cells in the process of apoptosis and cells that have just completed the apoptosis program. The proportion of cells undergoing apoptosis can be measured, for example, by detection of caspase cleavage or TUNEL staining. In some embodiments, less than 5% of the fibroblasts in the solution, such as less than about 4%, 3%, 2%, 1%, 0.5%, or 0.1% (including the range between any two of the recited values, such as 0.1%-5%, 0.1%-3%, 0.1%-1%, 0.5%-5%, 0.5%-3%, 0.5%-1%, 1%-5%, 1%-3%, or 3%-5%) are undergoing apoptosis. In some embodiments, less than about 1% of the fibroblasts in the solution are undergoing apoptosis.

[0096] It is noted that the methods, compositions, and kits according to some embodiments of the present specification can produce a large amount of mature ECM. In some embodiments, structures containing ECM are produced that are large enough to be visible under a microscope. In some embodiments, the solution contains nanostructures containing soluble ECM, and the nanostructures have a maximum diameter of at least 200 nm and up to 10,000 nm, for example at least 200 nm, 300 nm, 400 nm, 500 nm, 1000 nm, 2000 nm, 5000 nm, or more. In some embodiments, the nanostructures have a maximum diameter of 200 nm to 10,000 nm. Considering these large diameters, it is noted that filtration through a specific filter is unlikely to succeed because the structures containing ECM can easily clog the filter and cause a significant loss of the recovered ECM. Therefore, in some embodiments, the steps of manufacturing, purifying, and / or isolating the ECM include purification that does not include filtration. In some embodiments, the step of manufacturing the ECM includes purification that does not include sterile filtration. In some embodiments, the step of manufacturing the ECM includes purification that does not include 0.1 μM filtration. In some embodiments, avoidance of filtration (or sterile filtration) such as 0.1 μΜ filtration can substantially increase the yield of the recovered mature ECM. In some embodiments, the step of isolating the ECM from fibroblasts is performed without a sterile filtration solution containing the ECM. In some embodiments, the step of manufacturing the ECM is performed without a sterile filtration solution.

[0097] Some embodiments include a solution comprising fibroblasts and a soluble ECM, wherein at least about 90% of the fibroblasts in the solution are in the G0 cell cycle stage and less than 1% of the fibroblasts in the solution are undergoing apoptosis. The solution may include nanostructures comprising the soluble ECM. The nanostructures may have a maximum diameter of 200 nm to 10,000 nm. In some embodiments, the serum content (v / v) in the solution is less than 0.1%, e.g., less than 0.1%, less than 0.05%, less than 0.01%, less than 0.005%, or less than 0.001% (ranges between any two of the recited values, e.g., including a serum content of 0.1% to 0.001%). In some embodiments the solution is serum-free (as used herein, a serum “free” solution may optionally contain a small amount of serum that does not significantly affect the cell culture for ECM production). In some embodiments the solution is manufactured according to any of the methods described above.

[0098] Additional embodiments Some embodiments also include systems and methods for the manufacture and distribution of products containing human ECM and ECM for cosmetic and therapeutic uses. In some embodiments, the methods and systems provide manufacturing and distribution efficiencies of: 1) efficient removal, at low cost, of most of the dextran microcarriers sufficient to recover and further process human ECM without interference from the microcarriers; 2) desired features for the marketing and commercial implementation of xeno-free products (including methods and systems for communicating such desired features to manufacturers and users); and 3) efficiency in making products from both soluble and insoluble fractions from a single manufacturing lot (which can be more resource efficient than using only one of these fractions to make the product). Thus, the methods and systems according to some embodiments herein can provide efficiency in the manufacture of ECM and ECM-containing products.

[0099] Additional options are described below: 1. A method of manufacturing an extracellular matrix, comprising: differentiating induced pluripotent stem cells (iPSCs) into producer fibroblasts; Culturing the producing fibroblasts, whereby the producing fibroblasts produce an extracellular matrix (ECM); Isolating ECM from the producing fibroblasts, thereby manufacturing the ECM; A method comprising the above steps. Option 1 method further comprising, before differentiating iPSC into producing fibroblasts, dedifferentiating progenitor fibroblasts to form iPSC. Option 1 or 2 method further comprising expanding iPSC before said differentiation. Option 2 or 3 method further comprising constructing an iPSC bank before said differentiation. Option 1 method, wherein the iPSC to be differentiated is derived from only a single donor. Option 1 - 5 method, wherein the step of culturing the producing fibroblasts is in normoxia. Option 1 - 6 method, wherein the step of culturing the producing fibroblasts does not include culturing mesenchymal stem cells (MSC). Option 1 - 7 method, wherein the progenitor fibroblasts include adult skin (biopsy) fibroblasts. Option 1 - 8 method, wherein the step of isolating ECM includes purifying the ECM, thereby manufacturing a composition that is at least about 80 w / w% ECM. 10. The step of purifying the ECM includes washing the ECM in an acidic buffer; contacting a solution containing the producing fibroblasts and the ECM with dextranase; Option 9 method comprising the above steps. Option 1 - 10 method, wherein the ECM contains collagen. Option 11 method, wherein about 90% (w / w / ) of the ECM is COL1, and about 10% is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof. 13. A method according to any one of options 2 to 12, wherein the progenitor fibroblasts are further contacted with a dedifferentiation factor, thereby dedifferentiating the progenitor fibroblasts into iPSCs. 14. A method according to any one of options 1 to 13, wherein the iPSCs are free of viral insertions encoding Oct family members, Sox family members, and Klf family members. 15. A method according to any one of options 1 to 13, wherein the iPSCs are footprint-free. 16. A method according to any one of options 1 to 15, wherein the method does not include any of embryonic stem (ES) cells, bone marrow multipotent stem cells, ES-derived MSCs, or non-pluripotent neonatal foreskin fibroblast cell lines. 17. A method according to any one of options 1 to 16, wherein the ECM comprises the C-terminal propeptide of COL1, or triple helix or non-reduced gamma-type fibrillar collagen, or both. 18. A method according to any one of options 1 to 17, further comprising contacting the producing fibroblasts with serum until the producing fibroblasts produce mature collagen, and then gradually reducing the amount of serum until there is at least a 95% reduction in serum concentration. 19. The method of option 18, wherein the gradual reduction occurs over at least about 5 days. 20. A kit for producing an ECM, comprising: a composition comprising human fibroblasts; a dedifferentiation factor; a fibroblast differentiation factor; and a kit comprising the same. 21. The kit of option 20, wherein all of the fibroblasts in the composition are from a single donor. 22. The kit of any one of options 20 to 21, further comprising a substrate such as a dextran microcarrier. 23. The kit of option 22, further comprising dextranase and DNAase. 24. A composition comprising at least 80% (w / w) extracellular matrix, wherein the extracellular matrix is produced by a method according to any one of options 1 to 14. 25. iPSC-derived fibroblasts producing a mature extracellular matrix; a dedifferentiation factor and a cell culture comprising the same, wherein at least 50% (w / w) of the composition comprises ECM. 26. A method for producing extracellular matrix (ECM), comprising: culturing the fibroblast and / or MSC on a substrate comprising at least two surfaces until at least 80% of the fibroblast and / or MSC arrest their cell cycle, wherein the fibroblast and / or MSC define a three-dimensional shape on at least two surfaces, and the culturing is serum-free and xenofree; subsequently contacting the fibroblast and / or MSC with serum for at least about two weeks, thereby producing a soluble mature ECM by the fibroblast and / or MSC, thereby producing a xenofree solution comprising the soluble mature ECM and the fibroblast or MSC; isolating the soluble mature ECM from the produced fibroblast, thereby producing an ECM that is a mature xenofree ECM; and a method comprising the steps of 27. A method for expanding a human pluripotent cell culture, the expansion being serum-free and xenofree, thereby producing a human pluripotent cell, and contacting the human pluripotent cell with a differentiation factor, thereby differentiating the human pluripotent cell into a fibroblast or MSC; the method of option 26, further comprising the steps of 28. The method according to any one of options 26 to 27, wherein the contact between the fibroblast and / or MSC and the serum is from about two weeks to about eight weeks. 29. The method according to any one of options 26 to 27, wherein the contact between the fibroblast and / or MSC and the serum is at least about eight weeks. 30. The method according to any one of options 27 to 29, wherein the human pluripotent cell comprises induced pluripotent stem cells (iPSCs). 31. The method of option 30, wherein the iPSCs are footprint-free. 32. The method according to any one of options 30 to 31, wherein the iPSCs are derived from a single donor. 33. A method according to any one of options 26 - 32, further comprising the step of manufacturing a cosmetic composition comprising a mature xenofree ECM. 34. A method according to any one of options 26 - 33, further comprising the step of contacting fibroblasts or MSCs with ascorbic acid for at least two weeks while contacting with serum. 35. A method according to any one of options 26 - 34, wherein at least two surfaces of said fibroblasts or MSCs define a three - dimensional shape and the fibroblasts or MSCs are not contacted with serum until at least 70% of the fibroblasts or MSCs have arrested their cell cycle. 36. A method according to any one of options 26 - 35, wherein the amount of serum is about 0.1% - 10% (v / v). 37. A method according to any one of options 26 - 36, wherein the amount of serum is about 1 - 2% (v / v). 38. A method according to any one of options 26 - 37, wherein the serum comprises clinical - grade fetal bovine serum, pooled human serum, or a combination thereof. 39. A method according to any one of options 26 - 38, wherein the human pluripotent cells are a cell line previously expanded using animal components. 40. A method according to any one of options 26 - 39, wherein the mature xenofree ECM comprises fibrillar collagen. 41. A method according to option 40, wherein the mature xenofree ECM comprises the C - terminal propeptide of COL1, or triple - helix or non - reduced gamma - type fibrillar collagen, or both. 42. The solution comprises at least 250 μg of collagen per 1 cm 2 of substrate. A method according to any one of options 26 - 41. 43. The manufactured mature xenofree ECM comprises at least 250 μg of collagen per 1 cm 2 of substrate. A method according to any one of options 26 - 42. 44. A method according to any one of options 27 - 43, wherein the pluripotent cells are from a single donor. 45. A method according to any one of options 26 - 44, further comprising the step of detecting the amount of mature ECM in the solution. 46. The method of any one of options 26 - 45, further comprising the step of collecting a certain amount of used culture medium from the solution and isolating soluble mature ECM from the used culture medium. 47. A solution comprising fibroblasts or MSCs, and the soluble mature ECM produced according to any one of options 26 - 45, wherein the solution is xenofree and the soluble mature ECM contains crosslinked collagen. 48. A method for producing extracellular matrix (ECM), comprising: providing fibroblasts in a medium containing a certain concentration of serum; gradually reducing the amount of serum in the medium containing fibroblasts until the medium contains a serum concentration of 5% or less; after the step of gradually reducing, culturing the fibroblasts for at least about 2 weeks, whereby the fibroblasts produce soluble ECM, thereby producing a solution containing fibroblasts and soluble ECM; isolating the soluble ECM from the fibroblasts, thereby producing ECM; and a method comprising the above steps. 49. The method of option 48, wherein the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is at least 0.7 times the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. 50. The method of option 49, wherein the amount of fibroblasts in the medium at the start of the step of gradually reducing the amount of serum is at least 0.9 times the amount of fibroblasts in the medium when the medium contains a serum concentration of 5% or less. 51. The method of any one of options 48 - 50, wherein the step of gradually reducing the amount of serum is performed without cell expansion or subculture. 52. The method of any one of options 48 - 51, wherein the serum is gradually reduced for at least about 5 days. 53. The method of any one of options 48 - 52, wherein at least about 90% of the fibroblasts in the solution are in the G0 cell cycle stage. 54. The method of any one of options 48 - 53, wherein less than 1% of the fibroblasts in the solution are undergoing apoptosis. 55. A method according to any one of options 48 - 54, wherein the solution is a nanostructure comprising soluble ECM and comprises a nanostructure having a maximum diameter of 200 nm to 10,000 nm. 56. A method according to any one of options 48 - 55, wherein the step of manufacturing the ECM is performed without sterile filtration (so as not to exclude the nanostructure). 57. A method according to any one of options 48 - 56, wherein the step of isolating the soluble ECM from fibroblasts is performed without sterile filtration. 58. A solution comprising fibroblasts and soluble ECM, wherein at least about 90% of the fibroblasts in the solution are in the G0 cell cycle stage, less than 1% of the fibroblasts in the solution are undergoing apoptosis, and the solution is a nanostructure comprising soluble ECM and comprises a nanostructure having a maximum diameter of 200 nm to 10,000 nm. 59. A solution according to option 58, wherein the soluble ECM is manufactured by a method according to any one of options 48 - 57. 60. A method according to any one of options 1 - 19, 26 - 47, or 48 - 57, wherein the step of isolating the ECM from fibroblasts (such as producing fibroblasts, etc.) comprises separating the ECM from a substrate or solid phase. 61. A method according to any one of options 1 - 19, 26 - 47, 48 - 57, or 60, wherein upon isolation from fibroblasts (such as producing fibroblasts, etc.), the ECM is separated from any substrate or solid phase (for example, not bound to any substrate or solid phase). 62. A method according to any one of options 1 - 19, 26 - 47, 48 - 57, or 60 - 61, wherein upon isolation from fibroblasts (such as producing fibroblasts, etc.), the isolated ECM is not digested by any protease and is thus intact. 63. At least 10 liters of fibroblast cells are produced, for example, at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 liters of fibroblast cell cultures (ranges between any two of the listed values, for example, 10 to 1000 liters, 10 to 500 liters, 10 to 200 liters, 50 to 1000 liters, 50 to 500 liters, 50 to 200 liters, 100 to 1000 liters, 100 to 500 liters, or 100 to 200 liters are included), and any one of the methods of Options 1 to 19, 26 to 47, or 60 to 62 is used. 64. At least 10 liters of fibroblast cells that produce ECM are cultured, and any one of the methods of Options 48 to 57, or 60 to 62 is used. 65. At least 50 g of ECM is produced, for example, at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 g of ECM (including ranges between any two of the listed values), and any one of the methods of Options 1 to 19, 26 to 47, or 60 to 64 is used. 66. At least 50 g of soluble ECM is produced, for example, at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 g of soluble ECM (including ranges between any two of the listed values), and any one of the methods of Options 48 to 57, or 60 to 64 is used.

Examples

[0100] (Example 1) Human fibroblasts are obtained from a skin biopsy of a single donor. To induce the fibroblasts into footprint-free iPSCs, Oct4, Sox2, and c-Myc mRNAs are contacted with the fibroblasts. The footprint-free iPSCs are expanded 30-fold and the iPSCs are banked. To induce a commercial-scale quantity of iPSC cells to differentiate into fibroblasts, connective tissue growth factor (CTGF) is contacted with a commercial-scale quantity of iPSCs. The fibroblasts are cultured in a medium containing a dextran microcarrier substrate and then transferred to the production of mature ECM. The ECM is insoluble under culture conditions. The dextran microcarriers are digested using dextranase. The remaining insoluble fraction containing the mature ECM is recovered. Thus, the insoluble fraction of the culture containing the mature ECM is isolated from the fibroblasts and soluble components. Therefore, mature ECM is produced and the mature ECM is suitable for use in medical products and cosmetics.

[0101] (Example 2) Using the small molecule cocktail "VC6TF" (V, VPA; C, CHIR99021 or CHIR; 6, 616452; T, tranylcypromine; F, forskolin), human fibroblasts are chemically dedifferentiated into footprint-free iPSCs. The iPSCs are expanded to commercial production scale and banked by cryopreservation in liquid nitrogen. A commercial quantity of iPSCs is recovered and differentiated into fibroblasts using CTGF. The fibroblasts are cultured in a serum-free, xenofree medium containing a dextran microcarrier substrate. When the fibroblasts reach sufficient density on two or more surfaces of the substrate, approximately 90% of the fibroblasts enter the G0 phase of the cell cycle. At this point, human serum pooled from expired blood is added to the fibroblast culture to a concentration of 2% serum (v / v). Ascorbic acid is added to the culture together with the serum. The cells are cultured in serum for an additional 8 weeks and the cells produce mature ECM. The mature ECM is isolated from the cell culture and then used to manufacture cosmetics.

[0102] (Example 3) As described in Example 1, human fibroblasts are obtained from a single donor human iPSC. The fibroblasts are cultured in 2% fetal bovine serum, and the fibroblasts produce mature ECM. Every day for 10 days, half of the serum-containing medium is replaced with serum-free medium. Therefore, 10 days after the replacement, the 2% serum is 10 reduced by half, and as a result, the culture is estimated to contain less than 0.002% serum. After completion of the gradual serum replacement, the fibroblasts are cultured for 6 weeks in a medium (containing less than 0.002% serum, estimated value). The soluble ECM is recovered from the used medium. Further, since large structures (200 nm to 10,000 nm in diameter) containing ECM are present in the medium, the ECM is isolated without sterile filtration.

[0103] In some embodiments, a method, use, or composition includes various steps or features that exist as a single step or feature (as opposed to multiple steps or features). For example, in one embodiment, the method includes a single administration of a flow modulator, or the composition includes a flow modulator for single use, or consists essentially of. The flow modulator can be present in a single administration unit effective to increase the flow (or decrease immune cell migration). The composition or use can include a single administration unit of a flow modulator effective to increase the flow (or inhibit the migration of immune cells) as described herein. Multiple features or components are provided in alternative embodiments. In some embodiments, a method, composition, or use includes one or more means for flow regulation. In some embodiments, the means includes a flow modulator.

[0104] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and are not intended to be limiting, and the true scope and spirit are indicated by the following claims. For each method described herein, the related compositions for use in the method are clearly contemplated, as are the use of the compositions in the method and, where applicable, the method of making a pharmaceutical for use in the method. For example, with respect to a method of increasing a flow comprising a flow modulator, the use of the flow modulator in increasing the flow by the method is contemplated, as is the method of making a pharmaceutical comprising the flow modulator for use in increasing the flow, and the flow modulator for use in the corresponding method is also contemplated.

[0105] Those skilled in the art will understand with respect to this and other processes and methods disclosed herein that the functions performed in the processes and methods may be carried out in a different order. Further, the steps and operations outlined are provided only as examples, and some of the steps and operations may be optional and may be combined into fewer steps and operations or expanded into additional steps and operations without detracting from the essence of the disclosed embodiments.

[0106] With respect to the use of substantially any plural and / or singular terms herein, those skilled in the art can convert from plural to singular and / or from singular to plural as appropriate to the context and / or application. Various singular / plural permutations may be explicitly set forth herein for clarity.

[0107] In general, as used herein and in particular in the appended claims (e.g., the body of the appended claims), terms are generally intended to be “open” terms (e.g., the term “including” should be construed as “including but not limited to”, the term “having” should be construed as “having at least”, the term “include” should be construed as “includes but is not limited to”, etc.). It will be understood by those skilled in the art that if a specific number of claim recitations is intended, such intent will be explicitly recited in the claim, and absent such recitation, no such intent exists. For example, for purposes of illustration, the following appended claims may contain the use of introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to mean that the introduction of a claim recitation by the indefinite article “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even if the same claim contains the introductory phrase “one or more” or “at least one” and the indefinite article “a” or “an” (e.g., “a” and / or “an” should be construed to mean “at least one” or “one or more”). The same applies to the use of definite articles used to introduce claim recitations. Further, it will be recognized by those skilled in the art that even if a specific number of introduced claim recitations is explicitly recited, such recitations should be construed to mean at least the recited number (e.g., a minimum recitation of “two recitations” without other qualifying language means at least two recitations, or two or more recitations). Further, in examples where traditional expressions similar to “at least one of A, B, and C, etc.” are used, generally such syntax is intended in the sense that those skilled in the art will understand the traditional expression (e.g., “having at least one of A, B, and C” "Stem" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc. In examples where traditional expressions similar to "at least one of A, B, or C, etc." are used, generally such syntax is intended in the sense that those skilled in the art will understand the traditional expression. For example, "a system having at least one of A, B, or C" includes, but is not limited to, systems having only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc. It will be further understood by those skilled in the art that any substantially disjunctive words and / or phrases representing two or more alternative terms, whether in the specification, claims, or drawings, are intended to contemplate the possibility of including one of the terms, the other of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B".

[0108] Furthermore, when a feature or aspect of the present disclosure is described with respect to a Markush group, those skilled in the art will recognize that the present disclosure thereby describes it also with respect to any individual member of the Markush group or a subgroup of the members.

[0109] As will be understood by those skilled in the art, for all purposes, for example with respect to providing a written description, all ranges disclosed herein include any and all possible sub-ranges and combinations of such sub-ranges. Any recited range can be readily recognized as fully describing and enabling the same range divided into at least two equal parts, three equal parts, four equal parts, five equal parts, six equal parts, etc. By way of non-limiting example, each range discussed herein can be readily divided into a lower third, a middle third, and an upper third, etc. Also, as will be understood by those skilled in the art, all words such as "maximum", "at least", etc. include the recited number and refer to ranges that can then be divided into the sub-ranges discussed above. For example, "about 5" is to be taken to include the number 5. Finally, as will be understood by those skilled in the art, ranges include each individual number. Thus, for example, a group having 1 to 3 cells refers to a group having 1, 2, or 3 cells. Similarly, a group having 1 to 5 cells refers to a group having 1, 2, 3, 4, or 5 cells, etc. Terms such as "approximately", "about", and "substantially", when preceding a number as used herein, include the recited number (e.g., about 10% = 10%) and also represent an amount close to the recited amount that still performs the desired function or achieves the desired result. For example, the terms "approximately", "about", and "substantially" can refer to amounts that are less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of the recited amount.

[0110] From the foregoing, it will be understood that various embodiments of the present disclosure are described herein for purposes of illustration and that various modifications can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, and the true scope and spirit are indicated by the following claims.

Claims

1. A method for producing an extracellular matrix, the method comprising: a) differentiating induced pluripotent stem cells (iPSCs) into a plurality of producing fibroblasts; b) culturing the producing fibroblasts, whereby the producing fibroblasts produce an extracellular matrix (ECM), the method comprising culturing the producing fibroblasts in a cell culture medium containing serum at a concentration of 0.1% to 20% (v / v); c) gradually reducing the concentration of serum over a period of at least 5 days such that the concentration of serum is reduced by at least 95%; and d) isolating the ECM from the producing fibroblasts, thereby producing the ECM. A method comprising the above steps.

2. The method according to claim 1, further comprising dedifferentiating at least one precursor fibroblast to form the iPSCs prior to the step of differentiating the iPSCs into the producing fibroblasts.

3. The method according to claim 1 or 2, further comprising expanding the iPSCs prior to the step of differentiating.

4. The method according to any one of claims 1 to 3, wherein the culturing of the producing fibroblasts is carried out in normoxia.

5. The method according to any one of claims 1 to 4, wherein the step of culturing the producing fibroblasts does not include culturing mesenchymal stem cells (MSCs).

6. The method according to any one of claims 1 to 5, wherein the method does not include differentiating any of embryonic stem (ES) cells, bone marrow multipotent stem cells, ES-derived MSCs, or non-pluripotent neonatal foreskin fibroblast cell lines.

7. The method according to claim 1, wherein the ECM comprises triple helix collagen, non-reducing gamma-type fibrillar collagen, or both triple helix collagen and non-reducing gamma-type fibrillar collagen.

8. The method according to claim 1, wherein the step of isolating the ECM comprises purifying the ECM to thereby produce a composition that is at least about 80% (w / w) ECM.

9. The method according to claim 8, wherein about 90% (w / w) of the ECM is COL1 and about 10% is selected from the group consisting of COL3, COL4, COL5, COL6, and any combination thereof.

10. The method according to claim 1, wherein the step of isolating the ECM comprises contacting the ECM with an acidic buffer.

11. The method according to claim 1, wherein the step of isolating comprises contacting the ECM with dextranase.

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